US20200000862A1 - Oncolytic virus therapy - Google Patents

Oncolytic virus therapy Download PDF

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US20200000862A1
US20200000862A1 US16/482,623 US201816482623A US2020000862A1 US 20200000862 A1 US20200000862 A1 US 20200000862A1 US 201816482623 A US201816482623 A US 201816482623A US 2020000862 A1 US2020000862 A1 US 2020000862A1
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cells
tumor
virus
cancer
certain embodiments
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Zong Sheng Guo
David Bartlett
Zuqiang Liu
Mathilde Feist
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University of Pittsburgh
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C—CHEMISTRY; METALLURGY
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    • C07K2319/00—Fusion polypeptide
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    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the presently disclosed subject matter relates to oncolytic viruses, armed oncolytic viruses encoding, in an expressible form, an immunomodulator molecule, and to compositions of, and methods of making and using said oncolytic viruses.
  • the presently disclosed subject matter also relates to tumor infiltrated T cells—induced by oncolytic virus (“OV-induced T cells”), and to methods of making and using said OV-induced T cells for adoptive T-cell therapy.
  • OV-induced T cells induced by oncolytic virus
  • Oncolytic viruses which selectively replicate in and kill cancer cells, exert their anti-cancer effects through a number of modalities (Bartlett D L et al., 2013, Molecular Cancer 12:103-120).
  • oncolytic viruses can attack a cancer's blood supply, leading to apoptosis and necrosis of infected as well as non-infected cells.
  • oncolytic viruses induce immunogenic cell death (ICD) of cancer cells, release and present danger signal molecules (signal 0), along with inflammatory cytokines, and cross-present tumor-associated antigens (TAAs) to na ⁇ ve T cells, leading to elicitation of anti-tumor immunity (Guo Z S et al., 2014, Front Oncol 4:74).
  • ICD immunogenic cell death
  • TAAs tumor-associated antigens
  • the potent oncolytic viruses not only elicit potent and systemic adaptive antitumor immunity, but also promote the trafficking of tumor-specific CD8+ T cells into the tumor tissues (Bartlett D L et al., 2013, Mol Cancer 12:103; Guo Z S et al., 2017, 8:555).
  • agents that modulate the immune system for example agents that increase anti-cancer immunity and/or decrease anti-virus immunity.
  • agents capable of such immunomodulation cytokines and the like—can have profound, potentially dangerous effects on a treated subject when systemically dispersed.
  • adoptive T cell therapy is used as part of an anti-cancer regimen employing oncolytic virus to promote an immune response against cancer cells, and the oncolytic virus is optionally administered with an immunomodulator.
  • the immunomodulator is linked to the oncolytic virus.
  • the presently disclosed subject matter provides a method for generating tumor infiltrating oncolytic-virus induced T cells, comprising:
  • the presently disclosed subject matter provides a method of treating a subject suffering from cancer, comprising administering to the subject one or more tumor infiltrating oncolytic-virus induced T cells disclosed herein.
  • the presently disclosed subject matter provides a method of treating a subject suffering from cancer, comprising:
  • the oncolytic virus is a vaccinia virus. In certain embodiments, the oncolytic virus is a recombinant vaccinia virus with an inactivating mutation of its thymidine kinase gene, vaccinia growth factor gene, or both. In certain embodiments, the oncolytic virus is a herpes simplex virus. In certain embodiments, the oncolytic virus is an adenovirus.
  • the tumor-infiltrated T cells are expanded ex vivo in the presence of IL-2 and IL-7.
  • expanding the tumor-infiltrated T cells comprises co-culturing the tumor-infiltrated T cells with dendritic cells and cancer cells.
  • expanding the tumor-infiltrated T cells comprises culturing the tumor-infiltrated T cells with cytokines and/or agents.
  • the cytokines and/or the agents comprise one or more of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, IFN-alpha, IFN-alpha2, IFN-beta, or IFN-gamma, TNF-alpha, TNF-beta, and GM-CSF.
  • expanding the tumor-infiltrated T cells comprises culturing the tumor-infiltrated T cells with IL-2, IL-7, and/or GSK3b.
  • the subject suffering from cancer is treated with a cancer therapy before transferring the tumor-infiltrated T cells to the subject suffering from cancer.
  • the subject suffering from cancer is further treated with a cancer therapy.
  • the subject suffering from cancer is further with one or more exogenous cytokine and/or agent.
  • the subject suffering from cancer is further provided with exogenous IL-2, after transferring the tumor-infiltrated T cells.
  • the tumor-infiltrated cells are transferred intraperitoneally or intratumorally.
  • the presently disclosed subject matter is directed to the use of an oncolytic virus for treating a subject having cancer.
  • the presently disclosed subject matter is directed to the use of an oncolytic virus for generating tumor infiltrating oncolytic-virus induced T cells. In certain embodiments, the presently disclosed subject matter is directed to the use of tumor infiltrating oncolytic-virus induced T cells for treating a subject having cancer.
  • the tumor infiltrating oncolytic-virus induced T cells are generated by a method comprising:
  • the presently disclosed subject matter provides for the oncolytic viruses, the induced tumor-infiltrated T cells, therapeutic compositions comprising said viruses and T cells, and methods of treating subjects suffering from conditions that would benefit from immunomodulation, including patients suffering from various cancers.
  • the presently disclosed subject matter provides an oncolytic virus encoding a membrane-associated protein comprising an immunomodulator molecule linked to an anchoring peptide. In certain embodiments, the presently disclosed subject matter provides an oncolytic virus comprising, in its genome, a nucleic acid encoding a membrane-associated protein comprising an immunomodulator molecule linked to an anchoring peptide.
  • the oncolytic virus is a vaccinia virus. In certain embodiments, the oncolytic virus is a recombinant vaccinia virus with an inactivating mutation of its thymidine kinase gene, vaccinia growth factor gene, or both. In certain embodiments, the nucleic acid encoding the immunomodulatory molecule linked to the anchoring peptide is operably linked to the p7.5 e/l promoter.
  • the anchoring peptide comprises a GPI-anchor acceptor peptide or a PD-L1 transmembrane domain.
  • the immunomodulator molecule is joined to the anchoring peptide via a linker peptide.
  • the linker is a flexible linker or a rigid linker.
  • the immunomodulator molecule is interleukin-2 and/or interferon-gamma, and/or tumor necrosis factor-alpha.
  • an oncolytic virus is administered as a therapeutic composition.
  • the presently disclosed subject matter provides a therapeutic composition comprising an oncolytic virus together with a physiologic buffer.
  • the therapeutic composition is in lyophilized form.
  • the presently disclosed subject matter provides a syringe comprising an effective amount of the therapeutic composition.
  • the presently disclosed subject matter provides a method of treating a subject suffering from a cancer, comprising administering, to the subject, an effective amount of an oncolytic virus encoding an immunomodulator molecule.
  • the immunomodulator molecule is interleukin-2.
  • the cancer is locally invasive.
  • the cancer is metastatic.
  • the oncolytic virus is administered intratumorally.
  • a therapeutically effective amount of an immunomodulatory agent is further administered to the subject suffering from a cancer.
  • the immunomodulatory agent is an anti-PD-1 or an anti-PD-L1 antibody.
  • an additional therapy is provided to the subject suffering from cancer.
  • FIG. 1A-D Anti-tumor immunity elicited by viral delivered IL-2.
  • A Experimental scheme.
  • B-D Percent survival of immunocompetent mice treated with either phosphate buffered saline (PBS; dotted line), unarmed vvDD virus (black line) or mIL-2 “armed” vvDD-mIL-2 (gray line) five days after inoculation with (B) MC38-luc colon cancer cells; (C) ID8-luc ovarian cancer cells; or (D) AB12-luc mesothelioma cells.
  • PBS phosphate buffered saline
  • mIL-2 “armed” vvDD-mIL-2 gray line
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells (MC38 colorectal cancer model in C57BL/6 (B6) mice) (B) or 3.5 ⁇ 10 6 ID8-luc (ID8 ovarian cancer model in B6 mice) (C) or BALB/c mice were intraperitoneally inoculated with 4 ⁇ 10 5 AB12-luc (AB12 mesothelioma model in BALB/c mice) (D), and treated with PBS, vvDD and vvDD-IL-2 5 days post tumor inoculation.
  • the survival of tumor-bearing mice was shown by Kaplan-Meier analysis.
  • the standard symbols for P values are, * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001; and **** P ⁇ 0.0001.
  • FIG. 2A-B The toxicity of vvDD-mIL-2 in a late stage murine tumor model: vvDD-mIL-2 elicits strong toxic effects when treating more advanced MC38 colon cancer model in B6 mice.
  • A Experimental scheme.
  • B Percent survival of mice treated with either phosphate buffered saline (PBS; dotted line), unarmed vvDD virus (black line) or mIL-2 “armed” vvDD-mIL-2 virus (gray line) nine days after inoculation with MC38-luc colon cancer cells.
  • MC38 tumor model is normally treated on day 5. However, when treated on day 9, the expression of secreted IL-2 caused fatal effects in tumor-bearing mice.
  • FIG. 3 Examples of oncolytic viruses armed with immune-stimulatory molecules for elicitation of potent adaptive antitumor immunity.
  • the examples include, but not limited to, vvDD-mIL-2-GPI and vvDD-IL-23.
  • A Diagram of insertion, in vvDD thymidine kinase (tk) gene, of p7.5 e/l viral promoter operably linked to murine IL-2-encoding nucleic acid fused to (G4S)3 linker and GPI anchor-encoding sequences, in virus denoted vvDD-mIL-2-GPI.
  • This construct also contains nucleic acid encoding yellow fluorescent protein (“yfp”) operably linked to the pSe/l promoter as a detectable marker.
  • yfp yellow fluorescent protein
  • FIG. 1 Diagram of insertion, in vvDD thymidine kinase (tk) gene, of p7.5 e/l viral promoter operably linked to murine IL-23p19-encoding nucleic acid fused to IRES linker and murine IL-23p40 anchor-encoding sequences, in virus denoted vvDD-mIL-23p19-IRES.
  • This construct also contains nucleic acid encoding yellow fluorescent protein (“yfp”) operably linked to the pSe/l promoter as a detectable marker.
  • FIG. 4A-B Flow cytometry data.
  • A B16 melanoma cells infected with either vvD virus (left-most panel), vvDD-mIL-2 virus (center panel) or vvDD-mIL-2-GPI virus (right-most panel).
  • B MC38 colon cancer cells infected with either vvD virus (left-most panel), vvDD-mIL-2 virus (center panel) or vvDD-mIL-2-GPI virus (right-most panel).
  • FIG. 5A-B The expression intensity of IL-2 on cell membrane (A) in B16 cells infected with vvDD-mIL-2 or vvDD-mIL-2-GPI; (B) in MC38-luc cells infected with vvDD-mIL-2 or vvDD-mIL-2-GPI.
  • FIG. 6A-B The oncolytic viruses expressing IL-12 display significant therapeutic effect in early colon cancer model (day 5).
  • A Experimental scheme.
  • B Percent survival of mice treated with either phosphate buffered saline (PBS; dotted line), vvDD virus (black line), vvDD-mIL-2 virus (thin black line that stays at 100 percent) or IL-2 “anchored” vvDD-mIL-2-GPI (gray line) 5 days after inoculation with MC38-luc colon cancer cells (experimental scheme in upper part of the figure).
  • FIG. 7A-B (A) Experimental scheme. (B) Treatment mortality of MC38-luc tumor-bearing mice treated with either vvDD-mIL-2 (left bar) or vvDD-mIL-2-GPI (right bar) nine days after inoculation with MC38-luc colon cancer cells.
  • FIG. 8A-C Adoptive transfer of tumor infiltrated T cells—induced by oncolytic virus (“OV-induced T cells”) (isolated from vvDD-IL-2 treated MC38 tumor-bearing mice and ex vivo expanded) led to significant therapeutic effects in syngeneic C57BL/6 mice bearing peritoneal MC38 tumor.
  • OV-induced T cells induced by oncolytic virus
  • FIG. 8A-C Live imaging of the mice with MC38-luc tumors at day 21 post treatment and (D) at day 28 post treatment.
  • FIG. 9 Schematic diagram of viral IL-2 variants.
  • vvDD-IL-2, vvDD-IL-2-FG, vvDD-IL-2-RG and vvDD-IL-2-FPTM were generated by homologous recombination of murine IL-2 variants into the tk locus of vaccinia viral genome, carrying secreted IL-2, IL-2-flexible linker (G 4 S) 3 -GPI anchor sequence amplified form human CD16b, IL-2-rigid linker A(EA 3 K) 4 AAA-GPI anchor sequence amplified form human CD16b, IL-2-flexible linker (G 4 S) 3 -murine PD-L1 transmembrane domain, respectively.
  • FIG. 10A-B Viral replication and cytotoxicity in vitro.
  • Tumor cells MC38-luc, B16 and AB12-luc were mock-infected or infected with indicated viruses.
  • the production of virus progeny from infected cancer cells at 48 h post infection was determined by plaque assay (A), or the viability of viral infected cells was determinate by MTS assay (B).
  • FIG. 11A-C The expression and antitumor effect of viral delivered IL-2.
  • Tumor cell MC38-luc (3 ⁇ 10 5 cells), B16 (2 ⁇ 10 5 cells) or AB12-luc (3 ⁇ 10 5 cells) were mock-infected or infected with vvDD, vvDD-IL-2, vvDD-IL-2-FG, vvDD-IL-2-RG and vvDD-IL-2-FPTM at MOI of 1.
  • the culture supernatants were harvested for measuring secreted IL-2 by ELISA or membrane-associated IL-2 by flow cytometry 24 hours post infection.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated with PBS, vvDD, vvDD-IL-2, vvDD-IL-2-FG, vvDD-IL-2-RG and vvDD-IL-2-FPTM at 2 ⁇ 10 8 PFU/mouse 5 days post tumor inoculation.
  • C B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated with PBS, vvDD, vvDD-IL-2, vvDD-IL-2-FG and vvDD-IL-2-RG at 2 ⁇ 10 8 PFU/mouse 9 days post tumor inoculation. The survival of tumor-bearing mice was shown by Kaplan-Meier analysis. The standard symbols for P values are, * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001; and **** P ⁇ 0.0001.
  • FIG. 12A-B Viral delivered IL-2 expression in tumor cells.
  • Tumor cells MC38-luc, B16, or AB12-luc were mock-infected or infected with vvDD, vvDD-IL-2, vvDD-IL-2-FG, vvDD-IL-2-RG and vvDD-IL-2-FPTM at MOI of 1.
  • the infected cells were harvested 24 h post infection for RNA purification. Purified total RNAs were subject to RT-qPCR for quantitative detection of A34R mRNA (viral gene) (A) or IL-2 (B).
  • FIG. 13 Systemic antitumor immunity elicited by viral IL-2 variants.
  • FIG. 14A-H The more immunosuppressive microenvironment in 9-day-tumor-bearing mice.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and tumor tissues were harvested 5 days or 9 days post tumor inoculation.
  • Primary tumors were photoed (A), weighed (B), and lysed to make single cells and stained to determine the presence of CD4 + Foxp3 + (C), G-MDSC (CD11b + Ly6G + Ly6C lo ) (D), CD4 + PD-1 + (E), CD8 + PD-1 + (C), CD3 ⁇ NK1.1 + (G), and PD-L1 + (H) cells in the tumor microenvironment.
  • FIG. 15A-C The elevated expression of immunosuppressive factors and edema in 9-day-tumor-bearing mice.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and mouse tissues were harvested 5 days or 9 days post tumor inoculation.
  • A Primary tumors were applied to extract RNA for RT-qPCR to determine the expression of immunosuppressive factors. Livers and kidneys were harvested, weighed and dried to measure water content to determine liver edema (B) and lung edema (C).
  • FIG. 16A-E Toxicity profile of viral delivered IL-2.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated with PBS, vvDD, vvDD-IL-2, vvDD-IL-2-FG and vvDD-IL-2-RG at 2 ⁇ 10 8 PFU/mouse 9 days post tumor inoculation. The mice died earlier than PBS-treated mice, generally within 7 days post viral treatment, were counted as IL-2-induced death. The treatment related mortality was shown in (A).
  • mice were sacrificed 4 to 5 days post treatment for collecting blood to measure IL-2 (B) and TNF- ⁇ (C) in sera, and for monitoring lung edema (D) and liver edema (E).
  • the standard symbols for P values are, * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001; and **** P ⁇ 0.0001.
  • FIG. 17A-D The toxicity profile of vaccinia virus delivered IL-2 in early-stage-tumor-model.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated with vvDD, vvDD-IL-2, vvDD-IL-2-FG, vvDD-IL-2-RG, or PBS 5 days post tumor inoculation.
  • the treated mice were sacrificed 8 days post treatment to harvest tissues to measure water content to determine edema in lung (A), liver (B) and kidney (C), or to harvest blood to measure IL-2 (D) in sera.
  • FIG. 18A-N Immune status change in tumor microenvironment post virus treatments.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated with PBS, vvDD, vvDD-IL-2-FG and vvDD-IL-2-RG at 2 ⁇ 10 8 PFU/mouse 9 days post tumor inoculation.
  • Tumor-bearing mice were sacrificed 5 days post treatment and primary tumors were collected and analyzed by flow cytometry to determine CD4 + Foxp3 ⁇ and CD8 + IFN- ⁇ + T cells (A, B), memory-phenotype T cells (CD8 + CD44 hi ) (C), Regulatory T cells (CD4 + Foxp3 + ) (D), CD8/Treg (E), or by RT-qPCR to determine IFN- ⁇ , Granzyme B, Perforin, TGF- ⁇ , CD105, VEGF and IL-10 (F-I).
  • B6 mice were inoculated with 5 ⁇ 10 5 MC38-luc cells i.p.
  • ⁇ -CD8 Ab 250 ⁇ g/injection
  • ⁇ -NK1.1 Ab 300 ⁇ g/injection
  • ⁇ -CD4 Ab 150 ⁇ g/injection
  • ⁇ -IFN- ⁇ Ab 200 ⁇ g/injection
  • FIG. 19A-D The memory-phenotype CD8 + T cells and NK cells in 9-day-tumor-bearing mice.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated as described in FIG. 18 .
  • Primary tumors and spleens were harvested 5 days post viral treatment and applied to make single cells and stained to determine CD8 + CD44 hi in spleen (A), CD3 ⁇ NK1.1 + in tumor (B) and spleen (C), CD8 + T cells in tumor (D).
  • FIG. 20 The elevated NKP46 expression in 9-day-tumor-bearing mice post viral treatment.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated as described in FIG. 18 .
  • Primary tumors were harvested and applied to extract RNA for RT-qPCR to determine the expression of NKP46.
  • FIG. 21A-B The antitumor efficacy of vvDD combined with PD-L1 blockade.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated as scheduled (A), and the overall survival was shown by Kaplan-Meier analysis (B).
  • FIG. 22A-E Viral treatment apt to the combination with immune checkpoint therapy.
  • B6 mice were inoculated with 5 ⁇ 10 5 MC38-luc cells and treated as described in FIG. 18 . Tumor-bearing mice were sacrificed 5 days post treatment and primary tumors were collected and analyzed by RT-qPCR to determine the expression of PD-1, PD-L1, and CTLA-4 (A-C).
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated with vvDD-IL-2-RG or PBS 9 days post tumor inoculation.
  • ⁇ -PD-1 Ab 200 ⁇ g/injection
  • ⁇ -PD-L1 Ab 200 ⁇ g/injection
  • ⁇ -CTLA-4 Ab 100 ⁇ g/injection
  • FIG. 23A-C The elevated exhausted T cells and PD-L1 + cells in 9-day-tumor-bearing mice post viral treatment.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated as described in FIG. 18 .
  • Primary tumors were harvested and applied to make single cells and stained to determine CD4 + PD-1 + (A) and CD8 + PD-1+(B) T cells, and PD-L1+ cells (C).
  • FIG. 24A-B The antitumor efficacy of vvDD-IL-2-FG combined immune checkpoint therapy.
  • B6 mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and treated with vvDD-IL-2-FG or PBS 9 days post tumor inoculation.
  • ⁇ -PD-1 Ab 200 ⁇ g/injection
  • ⁇ -PD-L1 Ab 200 ⁇ g/injection
  • ⁇ -CTLA-4 Ab 100 ⁇ g/injection
  • FIG. 25A-C CD8 + T cell response in the tumor microenvironment (TME) of MC38 s.c. tumors 10 days post virus treatment.
  • TAE tumor microenvironment
  • CD8 + T cells have been analyzed for IFN- ⁇ production by ELISPOT.
  • CD8 + T cells (2 ⁇ 10 4 per well) were either left unstimulated (control) or challenged with y-irradiated MC38 tumor cells (2 ⁇ 10 4 per well) for 24 h. Results are shown as individual data points (number of spots in each well) and bars (means ⁇ standard deviation) of CD8 + T cells from each mouse evaluated in triplicates. To determine MC38 reactive responses the average value of spots from control wells were subtracted from the number of spots in MC38 challenged wells. Data were presented as individuals and means. Student's t-test was used to analyze the statistical significance (*p ⁇ 0.05).
  • CD8 + T cells in the TME are tumor specific.
  • B6 mice were s.c. inoculated with 5 ⁇ 10 5 MC38 cancer cells.
  • CD8 + T cells have been analyzed for IFN- ⁇ production by ELISPOT.
  • CD8 + T cells (2 ⁇ 10 4 per well) were either left unstimulated (medium control) or challenged with y-irradiated MC38 tumor cells (2 ⁇ 10 4 per well) or irrelevant target cells as y-irradiated B16 tumor cells (2 ⁇ 10 4 per well) or na ⁇ ve splenocytes (2 ⁇ 10 4 per well) from non-tumor-bearing B6 mouse for 24 h. Results are shown as individual data points (number of spots in each well) and bars (means ⁇ standard deviation) of CD8+ T cells from each mouse evaluated in duplicate. Student's t-test was used to analyze the statistical significance (*p ⁇ 0.05).
  • FIG. 26A-B CD8 + T cell response in the TME of MC38 s.c. tumors induced by vvDD-IL-2 is superior to IL-2 treatment.
  • CD8 + T cells have been analyzed for IFN- ⁇ production by ELISPOT.
  • CD8 + T cells (2 ⁇ 10 4 per well) were either left unstimulated (control) or challenged with ⁇ -irradiated MC38 tumor cells (2 ⁇ 10 4 per well) for 24 h. Results are shown as individual data points (number of spots in each well) and bars (means ⁇ standard deviation) of CD8 + T cells from each mouse evaluated in triplicates. To determine tumor reactive responses the average value of spots from control wells were subtracted from the number of spots in MC38 challenged wells. Student's t-test was used to analyze the statistical significance (*p ⁇ 0.05).
  • B For analysis of the systemic immune response additionally the spleen from each mouse has been harvested and proceeded to single cell suspension. Splenocytes have been analyzed for IFN- ⁇ production by ELISPOT.
  • Splenocytes (1 ⁇ 10 5 per well) were either left unstimulated (control) or challenged with ⁇ -irradiated MC38 tumor cells (2 ⁇ 10 4 per well) for 24 h. Results are shown as individual data points (number of spots in each well) and bars (means ⁇ standard deviation) of splenocytes from each mouse evaluated in triplicates. To determine tumor reactive responses the average value of spots from control wells were subtracted from the number of spots in MC38 challenged wells. Student's t-test was used to analyze the statistical significance (*p ⁇ 0.05).
  • FIG. 27A-B vvDD-IL-2 and vvDD treatment increase the number of tumor infiltrating T cells.
  • B Summary of the percentage of CD3+ T cells and CD3+CD4+ and CD3+CD8+ T cells per area.
  • FIG. 28 vvDD-IL-2 and vvDD treatment increases the number of T cells in the tumor.
  • B6 mice were s.c. inoculated with 5 ⁇ 10 5 MC38 cancer cells.
  • FIG. 29A-C Tumor Infiltrated T Cells—Induced by Oncolytic Virus (“OV-induced T cells”) can be cultured ex vivo and keep their tumor specificity.
  • OV-induced T cells Tumor Infiltrated T Cells—Induced by Oncolytic Virus (“OV-induced T cells”) can be cultured ex vivo and keep their tumor specificity.
  • T cells from each individual mouse have been cultured in 24-well plates (1 ⁇ 10 6 per well) in RPMI complete media in the presence of IL-2 (30 IU/ml) and IL-7 (5 ng/ml).
  • IL-2 (30 IU/ml)
  • IL-7 (5 ng/ml).
  • T cells (2 ⁇ 10 4 per well) were either left unstimulated (medium) or challenged with ⁇ -irradiated MC38 tumor cells (2 ⁇ 10 4 per well) or irrelevant target cells as ⁇ -irradiated B16 tumor cells (2 ⁇ 10 4 per well) or na ⁇ ve splenocytes (2 ⁇ 10 4 per well) from non-tumor-bearing B6 mouse in duplicate for 24 h. Results are shown as individual data points (number of spots in each well) and bars (means ⁇ standard deviation) of T cells from each mouse evaluated in duplicate. Student's t-test was used to analyze the statistical significance (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001).
  • T cells Besides IFN- ⁇ ELISPOT the T cells have been tested for 4-1BB upregulation by flow cytometry.
  • T cells (2 ⁇ 10 4 per well) were either left unstimulated (medium control) or challenged with ⁇ -irradiated MC38 tumor cells (2 ⁇ 10 4 per well) or irrelevant target cells as ⁇ -irradiated B16 tumor cells (2 ⁇ 10 4 per well) or na ⁇ ve splenocytes (2 ⁇ 10 4 per well) from non-tumor-bearing B6 mouse in duplicate. After 24 h the cells have been stained for flow cytometry analysis against CD3, CD4, CD8, 4-1BB.
  • Results are shown as individual data points (percentage of CD8 + 4-1BB + T cells and CD4 + 4-1BB + T cells) and bars (means ⁇ standard deviation) of T cells from each mouse evaluated in duplicate. Student's t-test was used to analyze the statistical significance (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001).
  • FIG. 30 Representative flow cytometry plot of CD8 + 4-1BB + and CD4 + 4-1BB + T cells of one sample from each group.
  • FIG. 31A-C In vitro analysis of the vvDD-IL-2 induced T cells versus na ⁇ ve T cells prior to adoptive T cell transfer. Prior to ACT the cultured T cells have been tested for tumor reactivity in vitro using IFN- ⁇ ELISPOT and co-culture assay for flow cytometry staining of 4-1BB expression and IFN- ⁇ ELISA.
  • T cells (2 ⁇ 10 4 per well) were either left unstimulated (medium) or challenged with ⁇ -irradiated MC38 tumor cells (2 ⁇ 10 4 per well) or irrelevant target cells as ⁇ -irradiated B16 tumor cells (2 ⁇ 10 4 per well) or na ⁇ ve splenocytes (2 ⁇ 10 4 per well) from non-tumor-bearing B6 mouse in duplicate for 24 h.
  • A ELISPOT Results are shown as individual data points (number of spots in each well) and bars (means ⁇ standard deviation) of T cells from each mouse evaluated in in duplicate.
  • B and C Besides IFN- ⁇ ELISPOT the T cells have been tested for 4-1BB Expression by flow cytometry.
  • the cells (2 ⁇ 10 4 per well) were either left unstimulated (medium control) or challenged with ⁇ -irradiated MC38 tumor cells (2 ⁇ 10 4 per well) or irrelevant target cells as ⁇ -irradiated B16 tumor cells (2 ⁇ 10 4 per well) or na ⁇ ve splenocytes (2 ⁇ 10 4 per well) from non-tumor-bearing B6 mouse in duplicate. After 24 h the cells have been stained for FLOW analysis against CD3, CD4, CD8, 4-1BB. Results are shown as individual data points (percentage of CD8 + 4-1BB + T cells and CD4 + 4-1BB + T cells) and bars (means ⁇ standard deviation) of T cells from each mouse evaluated in duplicate. Student's t-test was used to analyze the statistical significance (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001).
  • FIG. 32 Representative flow cytometry plot of CD8 + 4-1BB + and CD4 + 4-1BB + T cells of one sample from each group.
  • FIG. 33A-C Adoptive T cell transfer of vvDD-IL-2 induced T cells into MC38 i.p. tumor bearing mouse.
  • T cells have been expanded in 24-well plates (1 ⁇ 10 6 per well) in RPMI complete media in the presence of IL-2 (30 IU/ml) and IL-7 (5 ng/ml).
  • IL-2 (30 IU/ml)
  • IL-7 5 ng/ml
  • the na ⁇ ve T cells have been cultured under the same conditions as the virus induced T cells.
  • T cells have been harvested for adoptive transfer (5 ⁇ 10 6 T cells per mouse) 4 days later.
  • Prior to T cell transfer treated mouse received 5 Gy of sublethal irradiation to mimic lymphodepletion similar to clinical protocols.
  • Grouped mouse were intraperitoneally injected with vvDD-IL-2 induced T cells, na ⁇ ve T cells or PBS. All treated mouse received exogenous cytokine support of IL-2 (100.000 IU/mouse i.p. for 3 days every 12 h).
  • B The survival of tumor-bearing mouse was monitored by Kaplan-Meier analysis.
  • C The tumor growth has been monitored by live animal imaging.
  • FIG. 34 Imaging Day 17 post ACT.
  • an “oncolytic virus” is a virus that exhibits increased replication in, and lysis of, cancer cells relative to comparable non-cancer cells; see, for example, Bartlett D L et al., 2013, Molecular Cancer 12:103-120; Kaufman H L et al., 2015, Nature Reviews Drug Discovery 14:642-662 and Chiocca EA and Rabkin S D, 2014, Cancer Immunol Res; 2; 295-300.
  • the oncolytic virus exhibits selective replication in cancer cells and less or essentially no replication in non-cancer cells.
  • less replication means that replication in cancer cells versus comparable non-cancer cells is at least about 30 percent greater, or at least about 50 percent greater, or at least about 80 percent greater.
  • the term “about” or “approximately,” as used herein, can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” can mean an acceptable error range for the particular value, such as ⁇ 10% of the value modified by the term “about.”
  • Non-limiting examples of oncolytic viruses include types of (i) adenovirus (“Ad”), for example hTERT-Ad; (ii) herpes simplex virus (“HSV”), for example G207, HSV-1716, T-VEC, and HSV-2 APK mutant; (iii) poxvirus, for example vaccinia virus, for example vSP and vvDD (tk ⁇ /vgf ⁇ ) (and see below); (iv) arbovirus; (v) paramyxovirus, for example, measles virus, mumps and Newcastle disease virus; (vi) rhabdovirus, for example, vesicular stomatitis virus; (vii) picornavirus, for example Coxsackie virus, Seneca Valley Virus, and polio virus; (viii) reovirus; (ix) parvovirus; and (x) recombinant/engineered versions of any one of the above.
  • Ad adenovirus
  • HSV
  • the oncolytic virus is an oncolytic virus that has been approved by the Food and Drug Administration (FDA) or is undergoing clinical trials.
  • the oncolytic virus can be Talimogene laherparepvec (ImlygicTM; Amgen, Inc.), also referred to as T-VEC.
  • the oncolytic virus can be pelareorep (Reolysin®; Oncolytics Biotech, Inc.).
  • the oncolytic virus can be DNX-2401 (DNAtrix Therapeutics).
  • the oncolytic virus can be H101 (Oncorine®; Shanghai Sunway Biotech Co., Ltd.).
  • the oncolytic virus can be pexastimogene devacirepvec (JX-594; SillaJen Inc.). In certain embodiments, the oncolytic virus can be CG0070 (Cold Genesys, Inc.). In certain embodiments, the oncolytic virus can be G47 ⁇ (Daiichi-Sankyo Company, Limited).
  • the oncolytic virus is a vaccinia virus.
  • the oncolytic virus is an engineered (also referred to as “recombinant”) vaccinia virus.
  • the virus is a recombinant vaccinia virus based on the Western Reserve (“WR”) strain of vaccinia, for example, the WR strain commercially available from the American Type Culture Collection as ATCC No. VR1354.
  • WR Western Reserve
  • Other vaccinia virus strains suitable for engineering include, but are not limited, to the Wyeth strain (ATCC VR-1536), the Lederle-Chorioallantoic strain (ATCC VR-325), and the CL strain (ATCC VR-117).
  • the oncolytic virus is an engineered vvDD vaccinia viral construct comprising, for example, a modified version of a virus described in U.S. Pat. Nos. 7,208,313, 8,506,947, and United States Patent Application Publications Nos. 2003/0031681 and 2007/0154458, McCart et al., 2001, Cancer Research 61:8751-8757 and/or Thorne S et al., 2007, J. Clin. Invest. 117:3350-3358, all of which are incorporated by reference herein in their entries.
  • the vaccinia virus can have deletions of the thymidine kinase (tk) and/or vaccinia growth factor (vgf) genes.
  • a vaccinia virus has an inactivating mutation in one or more gene where the product of said gene or genes functions in viral replication.
  • one or more of the following genes can bear an inactivating mutation: the gene encoding the ribonucleotide reductase-large subunit, the gene encoding the ribonucleotide reductase-small subunit, the gene encoding thymidylate kinase, the gene encoding DNA ligase, the gene encoding dUTPase, the tk gene, and the vaccinia virus growth factor (vgf) gene.
  • an inactivating mutation is a mutation that either reduces or eliminates activity of the gene product.
  • gene activation can be achieved by mutagenesis, e.g., site-directed mutagenesis or PCR-mediated mutagenesis.
  • a nucleic acid can be inserted into one or more of the foregoing genes to achieve inactivation.
  • a nucleic acid encoding a protein can be inserted into one or more of the foregoing genes to achieve inactivation and to further achieve expression of the nucleic acid.
  • a nucleic acid encoding an immunomodulator molecule can be inserted within one of the foregoing genes to achieve inactivation.
  • a nucleic acid encoding an immunomodulator molecule linked to an anchoring peptide e.g., an ANCHIM protein
  • an anchoring peptide e.g., an ANCHIM protein
  • the oncolytic virus is a vaccinia virus having an inactivating mutation (a mutation that either reduces or eliminates activity of the gene product) in the tk gene.
  • the inactivation of the thymidine kinase gene can be generated by the insertion of a cytosine deaminase (fcy1) gene within the thymidine kinase gene locus of the vaccinia viral genome, resulting in the expression of the fcy1 gene rather than the tk gene.
  • a nucleic acid encoding a detectable protein for example a fluorescent protein, for example yellow fluorescent protein (“yfp”)
  • a nucleic acid encoding an immunomodulator can be inserted into the tk gene.
  • a nucleic acid encoding an ANCHIM protein can be inserted into the tk gene.
  • the recombinant vaccinia virus can have an inactivating mutation in the vaccinia growth factor gene.
  • a nucleic acid encoding an immunomodulator can be inserted into the vgf gene.
  • a nucleic acid encoding an ANCHIM protein can be inserted into the vgf gene.
  • immunomodulator molecules Any immunomodulator molecule known in the art can be utilized according to the presently disclosed subject matter. Certain cytokines have immunomodulatory activity and would be considered immunomodulator molecules (or, simply, “immunomodulators”) herein.
  • an immunomodulator molecule can comprise a cytokine. In certain embodiments, an immunomodulator molecule can comprise a chemokine.
  • immunomodulator molecules which can be used according to the presently disclosed subject matter include an interleukin (“IL”), for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24 or IL-27, C—X—C motif chemokine 11 (CXCL11), Chemokine (C-C motif) ligand 5 (CCL5), an interferon (“IFN”), for example, IFN-alpha, IFN-alpha2, IFN-beta, or IFN-gamma; or a tumor necrosis factor (“TNF”), for example TNF-alpha or TNF-be
  • IFN interferon
  • the immunomodulator molecule can be a human or non-human immunomodulator molecule.
  • Nucleic acid sequences encoding such immunomodulator molecules, and the encoded protein sequences, are well known in the art. Examples of non-human species include non-human primates, rodents, rabbits, dogs, cats, horses, pigs, sheep, cows, etc.
  • the immunomodulator molecule for use in the presently disclosed subject matter is IL-2.
  • human IL-2 comprises at least an immune-activating portion of the sequence APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:13; GenBank Acc. No. CAA25742.1, residues 21-153), or conservative substitutions thereof, e.g., said sequence having one or two amino acid variations.
  • the immunomodulator molecule for use in the presently disclosed subject matter is TNF-alpha.
  • the human TNF-alpha comprises at least an immune-activating portion of the sequence MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLF SFLIVAGATTLFCLLHFGVI GPQREEFPRDLSLISPLAQAVRS S SRTPSDKPVAHVVANPQAEGQLQWLNRRANA LLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTK VNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAE SGQVYFGIIAL (SEQ ID NO:15; NCBI Ref. Sequence No. NM_000594.3) or conservative substitutions thereof, e.g., said sequence having one or two amino acid variations.
  • the immunomodulator molecule for use in the presently disclosed subject matter is IL-23, which consists of the two subunits, IL-23A and IL-12B.
  • the human IL-23A protein comprises at least an immune-activating portion of the sequence MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVG HMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSD IFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRF KILRSLQAFVAVAARVFAHGAATLSP (SEQ ID NO:18; GenBank Acc. No. XXX, residues 1-189 or amino acid residues 28-184 of SEQ ID NO:18), or said sequence having one amino acid variation.
  • the presently disclosed subject matter can also be applied using a non-immunomodulator cytokine where said cytokine is desirably localized to the cancer cell environment, for example where said cytokine exhibits toxic effects when systemically administered.
  • conservative amino acid substitutions and “conservative modifications” refer to amino acid modifications that do not significantly affect or alter the function and/or activity of the presently disclosed proteins comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the proteins of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity.
  • amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a specified sequence are altered. Exemplary conservative amino acid substitutions are shown in Table 1 below.
  • the presently disclosed subject matter provides for an oncolytic virus that comprises a nucleic acid that encodes an immunomodulator molecule as set forth above.
  • the immunomodulator molecule encoding by the nucleic acid can be secreted, e.g., secreted from the cell infected by the oncolytic virus that comprises the nucleic acid.
  • the immunomodulator molecule can be linked to an anchoring peptide to become membrane-associated.
  • an oncolytic virus that comprises a nucleic acid that encodes an immunomodulator molecule that is to be secreted, e.g., secreted from the cell infected by the oncolytic virus that comprises the nucleic acid.
  • an oncolytic virus and immunomodulator molecules are discussed in the sections above.
  • the immunomodulator molecule can be of the same species as the subject intended to be treated (for example a human immunomodulator molecule to treat a human subject) or can be of a different species (for example a mouse immunomodulator molecule to treat a human subject).
  • the presently disclosed subject matter provides for an oncolytic virus, comprising, in its genome, a nucleic acid, encoding an immunomodulator molecule.
  • the oncolytic virus is a herpes simplex virus, a vaccinia virus, an adenovirus or a vesicular stomatitis virus.
  • the immunomodulator molecule can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, CXCL11, CCL5, an IFN, IFN-alpha, IFN-alpha2, IFN-beta, IFN-gamma, a TNF, TNF-alpha, TNF-beta, GM-CSF or a combination thereof.
  • the immunomodulator molecule is IL-2.
  • the immunomodulator molecule is IL-23.
  • the immunomodulator molecule is TNF-alpha.
  • the nucleic acid encoding the immunomodulator molecule can be placed under the control of a promoter that is active or activatable in an oncolytic virus infected cell, for example, a promoter of the oncolytic virus.
  • the encoding nucleic acid can be DNA, RNA or cDNA to conform to the nucleic acid of the viral genome into which it is inserted.
  • the oncolytic virus is a herpes simplex virus.
  • the herpes virus comprises, in its genome, a nucleic acid that encodes an immunomodulator molecule, as described above.
  • the nucleic acid encoding the immunomodulator molecule can be placed under the control of a promoter that is active or activatable in a herpes simplex virus infected cell, for example, a promoter of the herpes simplex virus.
  • the encoding nucleic acid can be DNA to conform to the nucleic acid of the genome of the herpes simplex virus.
  • an oncolytic virus of the present disclosure can be a herpes simplex virus that comprises a nucleic acid that encodes IL-2. In certain embodiments, an oncolytic virus of the present disclosure can be a herpes simplex virus that comprises a nucleic acid that encodes IL-23. In certain embodiments, an oncolytic virus of the present disclosure can be a herpes simplex virus that comprises a nucleic acid that encodes TNF-alpha.
  • the oncolytic virus is a vaccinia virus.
  • the nucleic acid encoding the immunomodulator molecule is operably linked to a promoter active or activatable in a vaccinia virus-infected cell, for example, a vaccinia virus promoter.
  • the nucleic acid encoding the immunomodulator molecule can be operably linked to a vaccinia promoter, for example the p7.5 e/l promoter, or the pSe/l promoter to generate a promoter/immunomodulator molecule-encoding construct.
  • the promoter/immunomodulator-encoding construct can be inserted into the tk gene.
  • an oncolytic virus of the present disclosure can be a vaccinia virus that comprises a nucleic acid that encodes IL-2. In certain embodiments, an oncolytic virus of the present disclosure can be a vaccinia virus that comprises a nucleic acid that encodes IL-23. In certain embodiments, an oncolytic virus of the present disclosure can be a vaccinia virus that comprises a nucleic acid that encodes TNF-alpha.
  • the presently disclosed subject matter provides for an oncolytic vaccinia virus, comprising, in its genome, a nucleic acid, which is a deoxyribose nucleic acid, encoding IL-2.
  • the nucleic acid can encode human IL-2 (e.g., comprising at least a portion of the amino acid sequence of SEQ ID NO:13 or conservative substitutions thereof).
  • the nucleic acid encoding IL-2 is operably linked to a promoter active or activatable in a vaccinia virus-infected cell, for example, a vaccinia virus promoter.
  • the nucleic acid encoding IL-2 can be operably linked to a vaccinia promoter, for example the p7.5 e/l promoter, or the pSe/l promoter to generate a promoter/IL-2-encoding construct.
  • the promoter/IL-2-encoding construct can be inserted into the tk gene.
  • the promoter/IL-2-encoding construct can be inserted into the vgf gene.
  • the presently disclosed subject matter provides for an oncolytic vaccinia virus, comprising, in its genome, a nucleic acid, which is a deoxyribose nucleic acid, encoding TNF-alpha.
  • the nucleic acid can encode human TNF-alpha (e.g., comprising the amino acid sequence of SEQ ID NO:15 or conservative substitutions thereof).
  • the nucleic acid encoding TNF-alpha is operably linked to a promoter active or activatable in a vaccinia virus-infected cell, for example a vaccinia virus promoter.
  • the nucleic acid encoding TNF-alpha can be operably linked to a vaccinia promoter, for example the p7.5 e/l promoter, or the pSe/l promoter to generate a promoter/TNF-alpha-encoding construct.
  • the promoter/TNF-alpha-encoding construct can be inserted into the tk gene.
  • the promoter/IL-2-encoding construct can be inserted into the vgf gene.
  • the presently disclosed subject matter provides for an oncolytic vaccinia virus, comprising, in its genome, a nucleic acid, which is a deoxyribose nucleic acid, encoding the subunits of IL-23, e.g., IL-23p19 and IL-23p40.
  • the nucleic acid encoding IL-23p19 and IL-23p40 is operably linked to a promoter active or activatable in a vaccinia virus-infected cell, for example, a vaccinia virus promoter.
  • the oncolytic vaccinia virus comprising, in its genome, a nucleic acid, which is a deoxyribose nucleic acid, that encodes a human homologue of IL-23p19, e.g., IL-23A, and a human homologue of IL-23p40, e.g., IL-12B, to generate IL-23.
  • a nucleic acid which is a deoxyribose nucleic acid
  • a human homologue of IL-23p40 e.g., IL-12B
  • the nucleic acid can encode IL-23p19 or a human homologue thereof, for example IL-23A, and/or IL-23p40 or a human homologue thereof, for example IL-12B, and can further be operably linked to a vaccinia promoter, for example the p7.5 e/1 promoter, or the pSe/l promoter to generate a promoter/IL-23p19/IL-23p40-encoding construct or a promoter/IL-23A/IL-12B-encoding construct.
  • a vaccinia promoter for example the p7.5 e/1 promoter
  • pSe/l promoter to generate a promoter/IL-23p19/IL-23p40-encoding construct or a promoter/IL-23A/IL-12B-encoding construct.
  • the promoter/IL-23p19/IL-23p40-encoding construct or a promoter/IL-23A/IL-12B-encoding construct can be inserted into the tk gene. In certain other non-limiting embodiments, the promoter/IL-23p19/IL-23p40-encoding construct or a promoter/IL-23A/IL-12B-encoding construct can be inserted into the vgf gene.
  • the presently disclosed subject matter provides for an oncolytic virus encoding a membrane-associated protein comprising an immunomodulator molecule as set forth above linked to an anchoring peptide, where the immunomodulator molecule linked to the anchoring peptide is referred to herein as the “ANCHIM”.
  • the membrane attachment domain is heterologous.
  • the membrane attachment domain is autologous. Suitable oncolytic viruses and immunomodulator molecules are discussed in the sections above. Anchoring peptides are discussed in the sections below.
  • the immunomodulator molecule can be of the same species as the subject intended to be treated (for example a human immunomodulator molecule to treat a human subject) or can be of a different species (for example a mouse immunomodulator molecule to treat a human subject).
  • the presently disclosed subject matter provides for an oncolytic virus, comprising, in its genome, a nucleic acid, encoding a membrane-associated protein comprising an immunomodulator molecule linked to an anchoring peptide.
  • the oncolytic virus is a herpes simplex virus, a vaccinia virus, an adenovirus or a vesicular stomatitis virus.
  • the immunomodulator molecule can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, CXCL11, CCL5, an IFN, IFN-alpha, IFN-alpha2, IFN-beta, IFN-gamma, a TNF, TNF-alpha, TNF-beta, GM-CSF or a combination thereof.
  • the immunomodulator molecule is IL-2.
  • the immunomodulator molecule is IL-23.
  • the immunomodulator molecule is TNF-alpha.
  • an anchoring peptide is any protein which, when fused to an immunomodulator molecule, anchors the immunomodulator molecule to the host cell membrane.
  • an anchoring peptide can be used according to the presently disclosed subject matter to modify an immunomodulator molecule to bind to the membrane of the host cell (e.g., by adding a GPI or other molecule).
  • the anchoring peptide is between about 10 and about 50 amino acids, or between about 15 and about 30, or about 20 amino acids in length.
  • the anchoring peptide is a glycosylphosphatidylinositol (GPI)-anchor acceptor peptide.
  • GPI anchor acceptor peptide comprises at least a portion of the C terminus of its native peptide, for example comprises at least a portion of the 100 C-terminal amino acids, or at least a portion of the 50 C-terminal amino acids, of the native peptide, or at least a portion of the 30 C-terminal amino acids.
  • the anchoring peptide can comprise sequences of various proteins as well as portions of cell membrane proteins that would serve essentially the same function.
  • the anchoring function can be achieved by a modification of an underlying peptide or protein that contains non-peptide elements, including but not limited to carbohydrate, lipid, etc.
  • the anchoring peptide can comprise a glycosylphosphatidylinositol (GPI)-anchor acceptor sequence of human CD16b anchor acceptor peptide.
  • the anchor is a GPI anchor acceptor peptide from a protein as set forth in Ferguson et al., “Chapter 11: Glycosylphosphatidyl Anchors” in Glycobiology, 2 nd Edition , Varki et al., editors, Cold Spring Harbor Press, 2009, for example but not limited to alkaline phosphatase, CD58, CD14, NCAM-120 and TAG-1, the contents of which are incorporated in their entirety by reference herein.
  • the GPI anchor acceptor peptide comprises a signal peptide portion (“SPP”), which functions during GPI addition and is cleaved in the process.
  • SPP signal peptide portion
  • a SPP comprises three domains: (1) a first domain comprising three relatively small amino acids (for example, but not limited to, Gly (G), Ala (A), Ser (S), Asn (N), Asp (D), or Cys (C) or any combination thereof) ⁇ , ( ⁇ +1), and ( ⁇ +2), where ⁇ is attached to the GPI anchor and ( ⁇ +1) and ( ⁇ +2) are the first 2 residues of the cleaved peptide; (2) a relatively polar domain spacer of about 5-10 amino acid residues and (3) a hydrophobic domain of about 15-20 amino acids.
  • the anchoring peptide can further comprise a sequence that targets it to the endoplasmic reticulum to facilitate addition of the GPI anchor if that is not a feature viral infection (see Mayor S and Riezman H, 2004, Nature Reviews Molecular Cell Biology 5, 110-120, the contents of which are incorporated in their entirety by reference herein.
  • This signal peptide portion extending from the protein C terminus, can be diagrammed as: ⁇ ( ⁇ +1) ⁇ ( ⁇ +2) ⁇ polar spacer region ⁇ ⁇ hydrophobic domain (see Galian C et al., 2012, J. Biol. Chem. 287(20):16399-16409).
  • the ⁇ ( ⁇ +1) ⁇ ( ⁇ +2) ⁇ polar spacer region can include a sequence of about ten amino acids of which at least about 2 or at least about 3 residues are G and at least about 2 or at least about 3 or at least about 4 or at least about 5 residues are S.
  • the sequence can be NSTGSGSSGS (SEQ ID NO:17; Galian, supra).
  • the hydrophobic domain can include between about 15 and about 20 amino acids comprising at least about 10 residues selected from the group of A, Leu (L), Val (V), Phe (F) and combinations thereof.
  • SPPs are provided in Table 1 or FIG. 8 of Galian, supra, and include:
  • the GPI anchor acceptor peptide sequence comprises any one of peptides having SEQ ID NO: 2-10 bearing about 1 or about 2 amino acid substitutions, insertions or deletions, or about 1, about 2 or about 3 conservative amino acid substitutions.
  • the GPI anchor acceptor peptide comprises the GPI anchor acceptor peptide sequence of CD16b comprising at least a portion, e.g., at least about 10 or at least about 20 or at least about 25 consecutive residues, of the sequence: SSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI (SEQ ID NO:1) or that sequence bearing about 1 or about 2 amino acid substitutions, insertions or deletions, or about 1, about 2 or about 3 conservative amino acid substitutions (see Simmons D and Seed B, 1988, Nature 333:568-570).
  • the GPI anchor acceptor peptide comprises the GPI anchor acceptor peptide sequence of CD16b comprising at least a portion, e.g., at least about 10 or at least about 20 or at least about 25 consecutive residues, of the sequence: VSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI (SEQ ID NO:11) or that sequence bearing about 1 or about 2 amino acid substitutions, insertions or deletions, or about 1, about 2 or about 3 conservative amino acid substitutions (see Simmons D and Seed B, 1988, Nature 333:568-570).
  • the anchoring peptide does not act via GPI.
  • the anchoring peptide can anchor its attached protein to a cell membrane via fatty acid/diacylglycerol.
  • the anchoring peptide comprises a region that can form a transmembrane domain, for example a substantially hydrophobic region and/or a region that is predicted to be a transmembrane domain by standard software, such as but not limited to TMpred.
  • the anchoring peptide is a PD-L1 transmembrane domain.
  • a nucleic acid encoding the immunomodulator molecule linked (optionally via a linker) to an anchoring peptide can be placed under the control of a promoter that is active or activatable in an oncolytic virus-infected cell, for example, a promoter of the oncolytic virus.
  • the encoding nucleic acid can be DNA, RNA, or cDNA to conform to the nucleic acid of the viral genome into which it is inserted.
  • the immunomodulator molecule can be linked directly to the anchoring peptide.
  • the immunomodulator molecule can be linked to the anchoring peptide via a linker.
  • the linker is selected from a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, a non-helical linker or a combination thereof.
  • the immunomodulator molecule can be linked to the anchoring peptide via one or more linkers, e.g., two or more, three or more or four or more.
  • the linker is a peptide linker.
  • the peptide linker comprises Gly and Ser.
  • the peptide linker can, for example and not by way of limitation, be between about 1 and about 25 or between about 5 and about 20 or between about 5 and about 15 amino acids in length.
  • Non-limiting examples of linkers for use in the presently disclosure subject matter are disclosed in International Patent Publication WO 2017/165464 (e.g., SEQ ID NOs:42, 44, 45, 75, 76, 77 and 78), the contents of which are incorporated by reference herein in its entirety.
  • the peptide linker comprises a flexible linker.
  • the flexible linker can be (G4S)3, which corresponds to GGGGSGGGGSGGGGS (SEQ ID NO:12).
  • the linker linked to the anchoring peptide can comprise the amino acid sequence GPAGGGGSGGGGSGGGGSVSTISSFSPPGYQVSFCLVMV LLFAVDTGLYFSVKTNI (SEQ ID NO:16) or that sequence bearing about 1 or about 2 amino acid substitutions, insertions or deletions, or about 1, about 2 or about 3 conservative amino acid substitutions.
  • GPI-anchored cytokines are provided in United States Patent Application Publication No. US 2003/0105054 (e.g., paragraphs [0045] and [0075]) and U.S. Pat. No. 6,277,368, the contents of which are hereby incorporated by reference in their entireties.
  • the peptide linker comprises a rigid linker.
  • the rigid linker can be (A(EA 3 K) 4 AAA) (SEQ ID NO: 36), which corresponds to AEAAAKEAAAKEAAAKEAAAKAAA.
  • the linker comprises the amino acid sequence SEQ ID NO:36 or that sequence bearing about 1 or about 2 amino acid substitutions, insertions or deletions, or about 1, about 2 or about 3 conservative amino acid substitutions.
  • the presently disclosed subject matter provides for an oncolytic virus encoding a membrane-associated protein comprising an immunomodulator molecule as set forth above linked to an anchoring peptide, optionally, via a linker.
  • the oncolytic virus is a herpes simplex virus, a vaccinia virus, an adenovirus or a vesicular stomatitis virus.
  • the immunomodulator molecule can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, CXCL11, CCL5, an IFN, IFN-alpha, IFN-alpha2, IFN-beta, IFN-gamma, a TNF, TNF-alpha, TNF-beta, GM-CSF or a combination thereof.
  • the immunomodulator molecule is IL-2.
  • the immunomodulator molecule is IL-23.
  • the immunomodulator molecule is IL-23.
  • the anchoring peptide comprises at least a portion of any one of SEQ ID NOs:1-11, for example said portion comprising between at least about 10 or at least about 20 or at least about 25 consecutive amino acid residues.
  • the anchoring peptide comprises the amino acid sequence of SEQ ID NO:11, or that sequence bearing about 1 or about 2 amino acid substitutions, insertions or deletions, or about 1, about 2 or about 3 conservative amino acid substitutions.
  • the immunomodulator molecule is joined to the anchoring peptide by a peptide linker between about 1 and about 25 or between about 5 and about 15 amino acids in length.
  • the linker comprises at least a portion of any one of SEQ ID NOs:12 and 36, for example said portion comprising between at least about 10 or at least about 20 or at least about 25 consecutive amino acid residues.
  • the linker comprises the sequence (G45)3 (SEQ ID NO:12) or conservative substitutions thereof.
  • the linker comprises the sequence (A(EA 3 K) 4 AAA) (SEQ ID NO: 36) or conservative substitutions thereof.
  • the oncolytic virus can comprise a gene encoding a membrane-associated fusion protein that includes an immunomodulator molecule fused to an anchoring peptide, e.g., a GPI anchor (e.g., a GPI-anchor acceptor sequence of human CD16b), via a rigid linker (alternatively referred to herein as “RGPI” or “RG”).
  • an anchoring peptide e.g., a GPI anchor (e.g., a GPI-anchor acceptor sequence of human CD16b), via a rigid linker (alternatively referred to herein as “RGPI” or “RG”).
  • the oncolytic virus can comprise a gene encoding a membrane-associated fusion protein that includes an immunomodulator molecule fused to an anchoring peptide, e.g., a GPI anchor (e.g., a GPI-anchor acceptor sequence of human CD16b), via a flexible linker (alternatively referred to herein as “FGPI” or “FG”).
  • an anchoring peptide e.g., a GPI anchor (e.g., a GPI-anchor acceptor sequence of human CD16b), via a flexible linker (alternatively referred to herein as “FGPI” or “FG”).
  • the GPI anchor sequence can be replaced with a PD-L1 transmembrane domain.
  • the oncolytic virus can comprise a gene encoding a membrane-associated fusion protein that includes an immunomodulator fused to a PD-L1 transmembrane domain, via a rigid linker. In certain embodiments, the oncolytic virus can comprise a gene encoding a membrane-associated fusion protein that includes an immunomodulator fused to a PD-L1 transmembrane domain, via a (G4S)3 linker. In certain embodiments, the oncolytic virus can comprise a gene encoding a membrane-associated fusion protein that includes an immunomodulator fused to a PD-L1 transmembrane domain, via a flexible linker (alternatively referred to herein as “FPTM”).
  • FPTM flexible linker
  • the presently disclosed subject matter provides for an oncolytic virus, e.g., a vaccinia virus, comprising, in its genome, a nucleic acid, which is a deoxyribose nucleic acid, encoding IL-2.
  • the IL-2 is human IL-2.
  • the human IL-2 comprises at least a portion of the amino acid sequence of SEQ ID NO:13 or conservative substitutions thereof, or said sequence having one amino acid or two amino acid variations.
  • the IL-2 is linked to an anchoring peptide, optionally with a linker peptide between IL-2 and the anchoring peptide.
  • the anchoring peptide comprises at least a portion of any one of SEQ ID NOS: 1-11, 17, and 37, for example said portion comprising between at least about 10 or at least about 20 or at least about 25 consecutive amino acid residues.
  • the IL-2 is joined to the anchoring peptide by a peptide linker between about 1 and about 25 or between about 5 and about 15 amino acids in length.
  • the nucleic acid encoding IL-2 is operably linked to a promoter active or activatable in an oncolytic virus-infected cell, for example, an oncolytic virus promoter (e.g., a vaccinia virus promoter).
  • the nucleic acid present in the genome of the oncolytic virus can encode human IL-2 (e.g., comprising the amino acid sequence of SEQ ID NO:13), linked to an anchoring peptide (e.g., comprising the amino acid sequence of SEQ ID NO:11) via a peptide linker (e.g., comprising the amino acid sequence of SEQ ID NO:12).
  • human IL-2 e.g., comprising the amino acid sequence of SEQ ID NO:13
  • an anchoring peptide e.g., comprising the amino acid sequence of SEQ ID NO:11
  • a peptide linker e.g., comprising the amino acid sequence of SEQ ID NO:12
  • the nucleic acid encodes APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLTGPAGGGGSGGGGSGGGGS VSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI (SEQ ID NO:14) or that sequence bearing about 1 or about 2 amino acid substitutions, insertions or deletions, or about 1, about 2 or about 3 conservative amino acid substitutions.
  • said encoding nucleic acid can be operably linked to an oncolytic virus promoter.
  • said encoding nucleic acid can be operably linked to a vaccinia promoter, for example the p7.5 e/l promoter, or the pSe/l promoter to generate a promoter/ANCHIM-encoding construct.
  • the promoter/ANCHIM-encoding construct can be inserted into the tk gene. In certain other non-limiting embodiments, the promoter/ANCHIM-encoding construct can be inserted into the vgf gene.
  • the nucleic acid can encode human IL-2 (e.g., comprising the amino acid sequence of SEQ ID NO:13), linked to an anchoring peptide (e.g., comprising the amino acid sequence of SEQ ID NOS:11 and 37) via a peptide linker (e.g., comprising the amino acid sequence of SEQ ID NO:12 and 36).
  • said encoding nucleic acid can be operably linked to a vaccinia promoter, for example the p7.5 e/l promoter, or the pSe/l promoter to generate a promoter/ANCHIM-encoding construct.
  • the promoter/ANCHIM-encoding construct can be inserted into the tk gene.
  • the promoter/ANCHIM-encoding construct can be inserted into the vgf gene.
  • the presently disclosed subject matter provides for an oncolytic vaccinia virus, comprising, in its genome, a nucleic acid, which is a deoxyribose nucleic acid, encoding TNF-alpha.
  • TNF-alpha is linked to an anchoring peptide, optionally with a linker peptide between TNF-alpha and the anchoring peptide.
  • the nucleic acid is operably linked to a promoter active or activatable in a vaccinia virus-infected cell, for example a vaccinia virus promoter.
  • the TNF-alpha is human TNF-alpha.
  • the human TNF-alpha comprises at least an immune-activating portion of SEQ ID NO:15, or said sequence having one or two amino acid variations.
  • the anchoring peptide comprises at least a portion of any one of SEQ ID NOS:1-11, for example said portion comprising at least between at least about 10 or at least about 20 or at least about 25 consecutive residues.
  • the anchoring peptide comprises SEQ ID NO:11, or that sequence bearing about 1 or about 2 amino acid substitutions, insertions or deletions, or about 1, about 2 or about 3 conservative amino acid substitutions.
  • the TNF-alpha is joined to the anchoring peptide by a peptide linker between about 1 and about 25 or between about 5 and about 15 amino acids in length.
  • the linker comprises the sequence (G45)3 (SEQ ID NO:12).
  • said encoding nucleic acid can be operably linked to a vaccinia promoter, for example the p7.5 e/l promoter, or the pSE/L promoter, to generate a promoter/ANCHIM-encoding construct.
  • the promoter/ANCHIM-encoding construct can be inserted into the tk gene.
  • the promoter/ANCHIM-encoding construct can be inserted into the vgf gene.
  • the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO:12).
  • the linker comprises the sequence RIGID LINKER (SEQ ID NO:36).
  • the presently disclosed subject matter provides for an oncolytic vaccinia virus, comprising, in its genome, a nucleic acid, which is a deoxyribose nucleic acid, encoding IL-23p19 and IL-23p40.
  • a nucleic acid which is a deoxyribose nucleic acid
  • the IL-23p19 and IL-23p40 are linked to an anchoring peptide, optionally with a linker peptide between IL-23p40 and the anchoring peptide.
  • the nucleic acid encoding IL-23p19 is operably linked to a promoter active or activatable in a vaccinia virus-infected cell, for example, a vaccinia virus promoter.
  • the oncolytic vaccinia virus comprising, in its genome, a nucleic acid, which is a deoxyribose nucleic acid, that encodes a human homologue of IL-23p19, e.g., IL-23A, and a human homologue of IL-23p40, e.g., IL-12B, to generate IL-23.
  • the human IL-23A protein comprises at least an immune-activating portion of the sequence SEQ ID NO:18 or said sequence having one amino acid variation.
  • the nucleic acid can encode IL-23p19 or a human homologue thereof, for example IL-23A (e.g., comprising the amino acid sequence of SEQ ID NO:18), and can further encode IL-23p40, or a human homologue thereof, for example IL-12B, linked to an anchoring peptide (e.g., comprising the amino acid sequence of SEQ ID NO:11 and 37) via a peptide linker (e.g., comprising the amino acid sequence of SEQ ID NO:12 and 36).
  • IL-23A e.g., comprising the amino acid sequence of SEQ ID NO:18
  • IL-12B a human homologue thereof, for example IL-12B
  • said encoding nucleic acid can be operably linked to a vaccinia promoter, for example the p7.5 e/l promoter, or the pSe/l promoter to generate a promoter/ANCHIM-encoding construct.
  • a vaccinia promoter for example the p7.5 e/l promoter, or the pSe/l promoter to generate a promoter/ANCHIM-encoding construct.
  • the promoter/ANCHIM-encoding construct can be inserted into the tk gene.
  • the promoter/ANCHIM-encoding construct can be inserted into the vgf gene.
  • the presently disclosed subject matter provides for an oncolytic herpes simplex virus, comprising, in its genome, a nucleic acid, encoding an immunomodulator molecule linked to an anchoring peptide, optionally with a linker peptide between the immunomodulator molecule and the anchoring peptide, as described above.
  • the nucleic acid encoding the immunomodulator molecule linked to an anchoring peptide, optionally with a linker peptide between the immunomodulator molecule and the anchoring peptide can be placed under the control of a promoter that is active or activatable in an oncolytic herpes simplex virus infected cell, for example, a promoter of the oncolytic herpes simplex virus.
  • the encoding nucleic acid linked to an anchoring peptide, optionally with a linker peptide between the immunomodulator molecule and the anchoring peptide can be DNA, RNA, or cDNA to conform to the nucleic acid of the viral genome into which it is inserted.
  • the presently disclosed subject matter further provides for pharmaceutical compositions comprising one or more of the above-described engineered (recombinant) oncolytic viruses, for example in a physiologic buffer, and for such therapeutic compositions in solid, liquid, frozen, or lyophilized form.
  • the presently disclosed subject matter further provides for a delivery device, for example a syringe, containing a therapeutically effective amount of such a pharmaceutical composition.
  • a delivery device for example a syringe
  • the oncolytic viruses disclosed herein can be administered according to any known method in the art.
  • a method for the delivery of an oncolytic virus, e.g., a vaccinia virus, as described herein or a pharmaceutical composition thereof or compositions that include anti-tumor T cells isolated from cancer tissue, to cancer or tumor cells can be via intratumoral injection.
  • alternate methods of administration can also be used, e.g., intravenous, via infusion, parenteral, intravenous, intradermal, intramuscular, transdermal, rectal, intraurethral, intravaginal, intranasal, intrathecal, or intraperitoneal.
  • the routes of administration can vary with the location and nature of the tumor.
  • the route of administration can be intradental, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., in the proximity of a tumor, particularly with the vasculature or adjacent vasculature of a tumor), percutaneous, intrathecal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, by lavage or orally.
  • the modified virus can be administered to the patient from a source implanted in the patient.
  • administration of the modified virus can occur by continuous infusion over a selected period of time.
  • an oncolytic vaccinia virus as described herein, or a pharmaceutical composition containing the same can be administered at a therapeutically effective dose by infusion over a period of about 15 mins, about 30 mins, about 45 mins, about 50 mins, about 55 mins, about 60 minutes, about 75 mins, about 90 mins, about 100 mins, or about 120 mins or longer.
  • the oncolytic vaccinia virus or the pharmaceutical composition of the present disclosure can be administered as a liquid dosage, wherein the total volume of administration is about 1 ml to about 5 ml, about 5 ml to 10 ml, about 15 ml to about 20 ml, about 25 ml to about 30 ml, about 30 ml to about 50 ml, about 50 ml to about 100 ml, about 100 ml to 150 ml, about 150 ml to about 200 ml, about 200 ml to about 250 ml, about 250 ml to about 300 ml, about 300 ml to about 350 ml, about 350 ml to about 400 ml, about 400 ml to about 450 ml, about 450 ml to 500 ml, about 500 ml to 750 ml or about 750 ml to 1000 ml.
  • a single dose of virus can refer to the amount administered to a subject or a tumor over a 1, 2, 5, 10, 15, 20 or 24 hour period.
  • the dose can be spread over time or by separate injection.
  • multiple doses (e.g., 2, 3, 4, 5, 6 or more doses) of the vaccinia virus can be administered to the subject, for example, where a second treatment can occur within 1, 2, 3, 4, 5, 6, 7 days or weeks of a first treatment.
  • multiple doses of the modified virus can be administered to the subject over a period of 1, 2, 3, 4, 5, 6, 7 or more days or weeks.
  • the oncolytic vaccinia virus or the pharmaceutical composition as described herein can be administered over a period of about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, about 10 weeks to about 11 weeks, about 11 weeks to about 12 weeks, about 12 weeks to about 24 weeks, about 24 weeks to about 48 weeks, about 48 weeks or about 52 weeks, or longer.
  • the frequency of administration of the oncolytic vaccinia virus or the pharmaceutical composition as described herein can be, in certain instances, once daily, twice daily, once every week, once every three weeks, once every four weeks (or once a month), once every 8 weeks (or once every 2 months), once every 12 weeks (or once every 3 months), or once every 24 weeks (once every 6 months).
  • therapeutically effective amount can refer to the amount of oncolytic virus that, when administered, can be sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated.
  • therapeuticically effective amount can also refer to the amount of oncolytic virus that is sufficient to elicit the biological or medical response of a cell, tissue, system, animal or human.
  • An effective amount in such method can include an amount that reduces growth rate or spread of the cancer or that prolongs survival in the subject.
  • This disclosure provides a method of reducing the growth of a tumor, which method can comprise administering, to the tumor, an effective amount of a modified virus as described above.
  • an effective amount of a modified virus, or a pharmaceutical composition thereof can include an amount sufficient to induce the slowing, inhibition or reduction in the growth or size of a tumor and can include the eradication of the tumor. Reducing the growth of a tumor can be manifested, for example, by reduced growth rate or a prolonged survival of a subject containing the tumor.
  • a “therapeutically effective amount” or “effective amount” can include an amount sufficient to induce the infiltration of T cells into the tumor and/or cancer.
  • an effective amount of virus can be determined by methods known in the art.
  • the virus can be administered in an amount sufficient to induce oncolysis in at least about 20% of cells in a tumor, in at least about 30% of cells in a tumor, in at least about 40% of cells in a tumor, in at least about 50% of cells in a tumor, in at least about 60% of cells in a tumor, in at least about 70% of cells in a tumor, in at least about 80% of cells in a tumor, or in at least about 90% of cells in a tumor.
  • the amount of virus administered can be between about 1 ⁇ 10 7 and 1 ⁇ 10 10 infectious viral particles or plaque forming units (pfu), or between about 1 ⁇ 10 7 and 1 ⁇ 10 9 pfu/m 2 surface area of the subject to be treated.
  • the virus can be administered at a dose that can comprise about 1 ⁇ 10 8 pfu.
  • the amount of virus administered can be between about 1 ⁇ 10 3 and 1 ⁇ 10 12 viral particles or pfu, or between about 1 ⁇ 10 5 and 1 ⁇ 10 10 pfu, or between about 1 ⁇ 10 5 and 1 ⁇ 10 8 pfu, or between about 1 ⁇ 10 8 and 1 ⁇ 10 10 pfu.
  • the virus can be administered at a dose that can comprise about 1 ⁇ 10 3 pfu/dose to about 1 ⁇ 10 4 pfu/dose, about 1 ⁇ 10 4 pfu/dose to about 1 ⁇ 10 5 pfu/dose, about 1 ⁇ 10 5 pfu/dose to about 1 ⁇ 10 6 pfu/dose, about 1 ⁇ 10 7 pfu/dose to about 1 ⁇ 10 8 pfu/dose, about 1 ⁇ 10 9 pfu/dose to about 1 ⁇ 10 10 pfu/dose, about 1 ⁇ 10 10 pfu/dose to about 1 ⁇ 10 11 pfu/dose, about 1 ⁇ 10 11 pfu/dose to about 1 ⁇ 10 12 pfu/dose, about 1 ⁇ 10 12 pfu/dose to about 1 ⁇ 10 13 pfu/dose, about 1 ⁇ 10 13 pfu/dose to about 1 ⁇ 10 14 pfu/dose, or about 1 ⁇ 10 14 pfu/dose to about 1 ⁇ 10 15 pfu
  • an oncolytic vaccinia virus of the presently disclosed subject matter can be administered at a dose that can comprise about 1 ⁇ 10 3 viral particles/dose to about 1 ⁇ 10 4 viral particles/dose, about 1 ⁇ 10 4 viral particles/dose to about 1 ⁇ 10 5 viral particles/dose, about 1 ⁇ 10 5 viral particles/dose to about 1 ⁇ 10 6 viral particles/dose, about 1 ⁇ 10 7 viral particles/dose to about 1 ⁇ 10 8 viral particles/dose, about 1 ⁇ 10 9 viral particles/dose to about 1 ⁇ 10 10 viral particles/dose, about 1 ⁇ 10 10 viral particles/dose to about 1 ⁇ 10 11 viral particles/dose, about 1 ⁇ 10 11 viral particles/dose to about 1 ⁇ 10 12 viral particles/dose, about 1 ⁇ 10 12 viral particles/dose to about 1 ⁇ 10 13 viral particles/dose, about 1 ⁇ 10 13 viral particles/dose to about 1 ⁇ 10 14 viral particles/dose, or about 1 ⁇ 10 14 viral particles/dose to about 1 ⁇ 10 15 viral particles/dose.
  • the presently disclosed subject matter relates to oncolytic viruses that promotes the infiltration of immune cells into the tumor microenvironment, thus becoming tumor-infiltrated immune cells induced by oncolytic virus.
  • the presently disclosed subject matter relates to oncolytic viruses that elicit systemic and potent anti-tumor immune cells, wherein the anti-tumor cells can be isolated from tumor tissues, expanded ex vivo, and administered to cancer patients for cancer treatments, e.g., in the mode of adoptive T cell transfer.
  • the presently disclosed subject matter relates to oncolytic viruses that promote the infiltration of T cells into the tumor microenvironment, thus becoming OV-induced T cells (also referred to herein as “tumor-infiltrated T cells” or “TILs”).
  • TILs OV-induced T cells
  • the oncolytic virus promotes infiltration of tumor-specific CD8+ and CD4+ T cells.
  • the oncolytic virus promotes infiltration of activated innate immune cells.
  • the activated innate immune cells comprise natural killer (NK) cells.
  • the presently disclosed subject matter relates to a method of making OV-induced T cells that comprises administering, to the subject, an effective amount of an oncolytic virus and inducing the elicitation and trafficking of T cells into the tumor tissue of the subject.
  • oncolytic viruses are disclosed above in section 5.1 and 5.3.
  • the oncolytic virus can be a vaccinia virus.
  • the vaccinia virus can be vvDD.
  • the oncolytic virus (e.g., vvDD) encodes, in expressible form, an immunomodulator molecule.
  • the immunomodulator molecule can be IL-2, IL-15, CXC11 and/or CCL5.
  • the presently disclosed subject matter relates to oncolytic vaccinia viruses (e.g., vvDD) encoding, in expressible form, at least one secreted and/or at least one membrane-associated immunomodulator molecule, as described above.
  • the oncolytic virus encodes, in an expressible form, IL-2, e.g., secreted or membrane-associated IL-2.
  • the method of making OV-induced T cells further comprises isolating OV-induced T cells from the subject, e.g., from the tumor of the subject.
  • OV-induced T cells can be isolated from lymphoid and non-lymphoid tissues or from peripheral blood.
  • OV-induced T cells can be isolated from cancer tissue.
  • the T cells can be isolated from tissue by digesting the tissue and using density gradient centrifugation, e.g., using a Percoll density gradient.
  • the method of making OV-induced T cells further comprises expanding the isolated OV-induced T cells ex vivo.
  • the isolated OV-induced T cells can be treated with one or more cytokines, lymphokines, and/or one or more agents, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, IFN-alpha, IFN-alpha2, IFN-beta, IFN-gamma, TNF-alpha, TNF-beta, GM-CSF or combinations thereof.
  • the isolated OV-induced T cells can be treated with IL-7, IL-2 and/or a GSK3b inhibitor.
  • the OV-induced T cells can be treated with IL-2.
  • the OV-induced T cells can be treated with IL-7.
  • the OV-induced T cells can be treated with IL-2 and IL-7.
  • the isolated T cells can be treated for about two to about 10 days, e.g., about three days.
  • the isolated OV-induced T cells are co-cultured with dendritic and cancer cells.
  • the OV-induced T cells can be co-cultured with cancer cells that have been infected with an oncolytic virus, e.g., an oncolytic virus disclosed herein.
  • the cancer cells can be infected with an oncolytic virus that is different than the oncolytic virus used to generate the OV-induced T cells.
  • the isolated OV-induce T cells are co-cultured with dendritic cells and cancer cells for about two to about 10 days, e.g., about two days.
  • the co-cultured T cells are then treated with one or more cytokines and/or one or more agents.
  • isolated OV-induced T cells can be analyzed with antibodies against CD3, CD8, CD4, and 4-1BB.
  • the oncolytic virus promotes an infiltration of CD3 + , CD8 + , CD4 + , and 4-1BB + T cells in the tumor microenvironment.
  • the isolated OV-induced T cells are CD3 + , CD8 + , CD4 + and/or 4-1BB + .
  • the OV-induced T cells are CD3 + .
  • the OV-induced T cells are CD3 + .
  • the OV-induced T cells are CD4 + .
  • the OV-induced T cells are CD8 + .
  • the OV-induced T cells are CD4 + 4-1BB + .
  • the OV-induced T cells are CD8 + 4-1BB + .
  • the OV-induced T cells can be used for cancer therapy via adoptive T cell transfer, as described in section 5.6.1. below.
  • the presently disclosed subject matter relates to adoptive immunotherapy, wherein the tumor-infiltrated T cells can be isolated from tumor tissues, expanded ex vivo, and administered to cancer patients for cancer treatments, e.g., in the mode of adoptive T cell transfer.
  • the OV-induced T cells can exert their functions to kill cancer cells and associated stromal cells.
  • the OV-induced T cells can be transferred to the subject from which the cells were isolated. Alternatively and/or additionally, the OV-induced T cells can be transferred to a different subject.
  • the OV-induced T cells are allogeneic.
  • the isolated OV-induced T cells prior to transfer of the isolated OV-induced T cells to a subject, can be analyzed for tumor specificity in a co-culture assay.
  • the isolated OV-induced T cells are co-cultured with target cancer cells, irrelevant cancer cells or splenocytes from for about one to about 10 days, e.g., about one day.
  • the cancer cells are ⁇ -irradiated.
  • the isolated OV-induced T cells are co-cultured with target cancer cells, irrelevant cancer cells or splenocytes.
  • the isolated OV-induced T cells for transfer can present specific reactivity against the target tumor cells.
  • the specific reactivity comprises an increase in the IFN- ⁇ expression by the isolated OV-induced T cells when they are co-cultured with target cancer cells, compared when co-cultured with irrelevant target cancer cells or splenocytes.
  • the present disclosure provides methods for the treatment of subjects that have cancer.
  • the present disclosure provides methods for the treatment of a subject that has cancer that includes administering isolated OV-induced T cells to the subject that has cancer as a mode of adoptive T cell immunotherapy.
  • the present disclosure further provides methods for the treatment of a subject that has cancer, which includes the administration of oncolytic viruses that are armed with an immunomodulator molecule, e.g., secreted or membrane associated.
  • Non-limiting examples of oncolytic viruses for use in the presently disclosed methods are disclosed above in sections 5.1, 5.3 and 5.5 above. Methods of administering oncolytic viruses is disclosed above in section 5.4 above.
  • a subject can be a human or a non-human subject.
  • Non-limiting examples of non-human subjects include non-human primates, dogs, cats, horses, pigs, cows, mice, rats, hamsters and other rodents, rabbits, etc.
  • Non-limiting examples of cancers that can be treated by the disclosed methods include gastrointestinal cancer, colon carcinoma, colorectal cancer, ovarian carcinoma, mesothelioma, melanoma, breast cancer, brain cancer (e.g., glioblastoma), prostate cancer, cervical cancer, non-small cell lung cancer, renal carcinoma, hepatic cancer, pancreatic cancer, biliopancreatic cancer, adenocarcinoma of the liver, gastric cancer, liver cancer, peritoneal cancer, pleural cancer, hematopoietic cancer, and metastatic cancer.
  • gastrointestinal cancer colon carcinoma, colorectal cancer, ovarian carcinoma, mesothelioma, melanoma, breast cancer, brain cancer (e.g., glioblastoma), prostate cancer, cervical cancer, non-small cell lung cancer, renal carcinoma, hepatic cancer, pancreatic cancer, biliopancreatic cancer, adenocarcinoma of the liver, gastric cancer, liver
  • treatment by administration of isolated OV-induced T cells or by using a modified virus can be used alone or in combination with one or immunomodulatory agents.
  • An immunomodulatory agent can include any compound, molecule or substance capable of suppressing antiviral immunity associated with a tumor or cancer.
  • the immunomodulatory agent can be capable of suppressing innate immunity or adaptive immunity to the modified virus.
  • Non-limiting examples of immunomodulatory agents include anti-CD33 antibody or variable region thereof, an anti-CD11b antibody or variable region thereof, a COX2 inhibitor, e.g., celecoxib, cytokines, such as IL-12, GM-CSF, IL-2, IFN3 and IFN ⁇ , and chemokines, such as MIP-1, MCP-1 and IL-8.
  • the immunomodulatory agent includes immune checkpoint inhibitors such as, but not limited to, anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PDL1 antibodies and TLR agonists (e.g., Poly I:C).
  • the checkpoint inhibitors can include molecules and/or compounds that inhibit and/or reduce PD-1, PD-L1 and/or CTLA4 activity and/or function.
  • the immunomodulatory agent can be administered systemically or locally.
  • “In combination with,” as used herein, means that a virus, such as an oncolytic vaccinia virus as described herein or a pharmaceutical composition thereof, and the additional therapy, are administered to a subject as part of a treatment regimen or plan.
  • being used in combination does not require that the oncolytic virus and the one or more agents are physically combined prior to administration or that they be administered over the same time frame.
  • the oncolytic virus and the one or more agents can be administered concurrently to the subject being treated, or can be administered at the same time or sequentially in any order or at different points in time.
  • methods of the present disclosure can include the administration of immunomodulatory agent prior to administration of an oncolytic virus, e.g., an oncolytic virus that comprises a nucleic acid that encodes an immunomodulatory molecule, e.g., IL-2.
  • an oncolytic virus e.g., an oncolytic virus that comprises a nucleic acid that encodes an immunomodulatory molecule, e.g., IL-2.
  • the immunomodulatory agent can be IL-2, e.g., administered systemically or locally.
  • the methods of the presently disclosed subject matter can include promoting an initial immune response in the subject to be treated by administering an oncolytic virus, disclosed herein, e.g., an oncolytic virus that comprises a nucleic acid that encodes an immunomodulatory molecule, e.g., IL-2, and/or the administration of immunomodulatory agent, e.g., IL-2.
  • an oncolytic virus disclosed herein, e.g., an oncolytic virus that comprises a nucleic acid that encodes an immunomodulatory molecule, e.g., IL-2, and/or the administration of immunomodulatory agent, e.g., IL-2.
  • the methods of the presently disclosed subject matter can comprise administering one or more isolated OV-induced T cells, a virus as disclosed herein or a pharmaceutical composition thereof, followed by, preceded by or in combination with one or more additional therapies.
  • Non-limiting examples of the such therapies can include chemotherapy, radiation, oncolytic viral therapy with an additional virus, treatment with immunomodulatory proteins, an anti-cancer agent or any combinations thereof.
  • Anti-cancer agents can include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, cytokines, immune checkpoint inhibitors, anti-angiogenic agents, apoptosis-inducing agents, anti-cancer antibodies and/or anti-cyclin-dependent kinase agents.
  • the cancer therapies can include chemotherapy, biological therapy, radiotherapy, immunotherapy, hormone therapy, anti-vascular therapy, cryotherapy, toxin therapy and/or surgery or combinations thereof.
  • Certain non-limiting embodiments of the presently disclosed subject matter provide for methods of treating cancer where the cancer is an early stage cancer. Certain non-limiting embodiments of the presently disclosed subject matter provide for methods of treating cancer as set forth above where the cancer is not an early stage cancer.
  • the cancer can be a late stage cancer.
  • late stage cancer can refer to a cancer that is no longer be limited to the organ of origin: e.g., it may have locally extended beyond the organ of origin or may have metastasized to another site in the body.
  • a late stage cancer can be a Grade III cancer.
  • the presently disclosed subject matter can be used to treat locally invasive or metastatic forms of cancer.
  • the presently disclosed subject matter can be applied to treatment of colon cancer that is stage T3 or higher and/or M1 or higher. In certain embodiments, the presently disclosed subject matter can be applied to treatment of ovarian cancer that is stage 1C or higher. In another certain non-limiting embodiment, the presently disclosed subject matter can be applied to the treatment of mesothelioma that is stage 2 or stage 3 or higher. In certain embodiments, the presently disclosed subject matter can be applied to the treatment of melanoma that is Stage III or higher.
  • the presently disclosed subject matter provides for methods of treating a subject suffering from a cancer with isolated OV-induced T cells.
  • the OV-induced T cells are produced as described above in section 5.5. above.
  • the OV-induced T cells can be transferred to the subject from which the cells were isolated. Alternatively and/or additionally, the OV-induced T cells can be transferred to a different subject.
  • the presently disclosed subject matter provides for an adoptive T-cell immunotherapy, comprising isolating the OV-induced T cells, expanding them ex vivo, and transferring them back to a subject suffering from a cancer.
  • Methods for adoptive T cell transfer are disclosed in US 2003/0170238, the contents of which are incorporated herein by reference.
  • the method of treating a subject suffering from a cancer comprises administering a therapeutically effective amount of OV-induced T cells to the subject.
  • the OV-induced T cells were isolated from a different subject.
  • the OV-induced T cells were isolated from the subject that is to be treated with the OV-induced T cells.
  • the method of treating a subject suffering from a cancer comprises administering, to the subject, an effective amount of an oncolytic virus, inducing the elicitation of potent antitumor T cells, promoting the trafficking of the induced potent antitumor T cells into the tumor tissues to generate OV-induced T cells, isolating the OV-induced T cells and administering the isolated OV-induced T cells to the subject.
  • the method of treating a subject suffering from a cancer comprises (a) administering, to the subject, an effective amount of an oncolytic virus; (b) isolating OV-induced T cells; (c) expanding the tumor-infiltrated T cells; and (d) transferring the tumor-infiltrated T cells to the subject suffering from cancer.
  • the isolated OV-induced T cells treated with one or more cytokines and/or one or more agents and expanded ex vivo, as described above, are then introduced, e.g., intraperitoneally (i.p.), into a cancer patient.
  • the isolated OV-induced T cells are administered intratumorally in a cancer patient.
  • the oncolytic virus can be a herpes simplex virus, a vaccinia virus, an adenovirus or a vesicular stomatitis virus.
  • the oncolytic virus can be the Western Reserve strain of vaccinia virus.
  • the oncolytic virus is the vvDD vaccinia virus.
  • the oncolytic virus is a vaccinia virus, e.g., vvDD.
  • the oncolytic virus can comprise, in its genome, a nucleic acid, encoding an immunomodulator molecule.
  • the presently disclosed subject matter relates to oncolytic vaccinia viruses encoding, in expressible form, at least one secreted and/or at least one membrane-associated immunomodulator molecule comprising an immunomodulator molecule linked to a membrane attachment domain, e.g., anchoring peptide, as described above.
  • the immunomodulator molecule can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, CXCL11, CCL5, an IFN, IFN-alpha, IFN-alpha2, IFN-beta, IFN-gamma, a TNF, TNF-alpha, TNF-beta, GM-CSF or a combination thereof.
  • the immunomodulator molecule is IL-2.
  • the immunomodulator molecule is IL-23. In certain embodiments, the immunomodulator molecule is TNF-alpha. In certain embodiments, the immunomodulator molecule is CXC11. In certain embodiments, the immunomodulator molecule is CCL5. In certain embodiments, the immunomodulator molecule is IL-15.
  • the oncolytic virus e.g., vaccinia virus
  • the oncolytic virus is the vvDD vaccinia virus that comprises a nucleic acid that encodes IL-2.
  • the oncolytic virus e.g., vaccinia virus
  • the oncolytic virus is a Western Reserve strain of vaccinia virus that comprises a nucleic acid that encodes IL-2 linked to an anchoring peptide.
  • the oncolytic virus is the vvDD vaccinia virus that comprises a nucleic acid that encodes IL-2 linked to an anchoring peptide.
  • the IL-2 is linked to the anchoring peptide via a rigid linker.
  • the oncolytic virus is a Western Reserve strain of vaccinia virus that comprises a nucleic acid that encodes IL-2 linked to an anchoring peptide via a rigid linker, e.g., a linker comprising the (A(EA 3 K) 4 AAA) linker.
  • the oncolytic virus is the vvDD vaccinia virus that comprises a nucleic acid that encodes IL-2 linked to an anchoring peptide via a rigid linker, e.g., a linker comprising the (A(EA 3 K) 4 AAA) linker.
  • cancer patients prior to the T cell transfer, can be administered another cancer therapy, disclosed above.
  • cancer patients after to the T cell transfer, cancer patients can be administered another cancer therapy, disclosed above.
  • the cancer therapies can include chemotherapy, biological therapy, radiotherapy, immunotherapy, hormone therapy, anti-vascular therapy, cryotherapy, toxin therapy and/or surgery or combinations thereof.
  • a cancer patient can be administered one or more exogenous cytokines and one or more agents, e.g., immunomodulatory agents, prior to the T-cell transfer.
  • a cancer patient can be administered one or more exogenous cytokines and one or more agents after the T-cell transfer.
  • the cytokines and/or agents can be locally or systemically administered. In certain embodiments, the cytokines and/or agents are locally administered.
  • a cancer patient can receive exogenous IL-2 before and/or after T cell transfer.
  • a cancer patient can receive exogenous IL-2 after T cell transfer.
  • the subject administered the OV-induced T cells of the presently disclosed subject matter can undergo radiation therapy prior and/or after to administration of the OV-induced T cells.
  • the radiation dose can be about 100 Rads (1 Gy) to about 500 Rads (5 Gy), about 5,000 Rads (50 Gy) to about 100,000 Rads (1000 Gy), or about 50,000 Rads (500 Gy), or other appropriate doses within the recited ranges.
  • “Gy” as used herein can refer to a unit for a specific absorbed dose of radiation equal to 100 Rads. Gy is the abbreviation for “Gray.”
  • the presently disclosed subject matter further provides for methods of treating a subject suffering from a cancer, comprising administering, to the subject, an effective amount of an oncolytic virus, e.g., an armed oncolytic virus, disclosed herein.
  • an oncolytic virus e.g., an armed oncolytic virus
  • Non-limiting examples of oncolytic viruses, e.g., armed oncolytic viruses, for use in the presently disclosed methods are disclosed above in sections 5.1., 5.3., and 5.5. above. Methods of administering oncolytic viruses is disclosed above in section 5.4 above.
  • the oncolytic virus can be a herpes simplex virus, a vaccinia virus, an adenovirus or a vesicular stomatitis virus that comprises, in its genome, a nucleic acid encoding an immunomodulator molecule as described herein.
  • the oncolytic virus can be the Western Reserve strain of vaccinia virus that comprises, in its genome, a nucleic acid encoding an immunomodulator molecule as described herein.
  • the oncolytic virus is the vvDD vaccinia virus that comprises, in its genome, a nucleic acid encoding an immunomodulator molecule as described herein.
  • the oncolytic encodes, in expressible form, at least one secreted immunomodulator molecule and/or at least one membrane-associated immunomodulator molecule, as described above.
  • the immunomodulator molecule can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, CXCL11, CCL5, an IFN, IFN-alpha, IFN-alpha2, IFN-beta, IFN-gamma, a TNF, TNF-alpha, TNF-beta, GM-CSF or a combination thereof.
  • the immunomodulator molecule is IL-2. In certain embodiments, the immunomodulator molecule is IL-23. In certain embodiments, the immunomodulator molecule is TNF-alpha. In certain embodiments, the immunomodulator molecule is CXC11. In certain embodiments, the immunomodulator molecule is CCL5. In certain embodiments, the immunomodulator molecule is IL-15.
  • the oncolytic virus e.g., vaccinia virus, can comprise a nucleic acid that encodes IL-2. In certain embodiments, the oncolytic virus is a Western Reserve strain of vaccinia virus that comprises a nucleic acid that encodes IL-2.
  • the vvDD vaccinia virus that comprises a nucleic acid that encodes IL-2.
  • the oncolytic virus e.g., vaccinia virus
  • the oncolytic virus is a Western Reserve strain of vaccinia virus that comprises a nucleic acid that encodes IL-2 linked to an anchoring peptide.
  • the IL-2 is linked to the anchoring peptide via a rigid linker.
  • the oncolytic virus is a Western Reserve strain of vaccinia virus that comprises a nucleic acid that encodes IL-2 linked to an anchoring peptide via a rigid linker, e.g., a linker comprising the (A(EA 3 K) 4 AAA) linker.
  • the oncolytic virus is the vvDD vaccinia virus that comprises a nucleic acid that encodes IL-2 linked to an anchoring peptide via a rigid linker, e.g., a linker comprising the (A(EA 3 K) 4 AAA) linker.
  • the presently disclosed subject matter provides for methods of treating a subject suffering from a cancer, comprising administering, to the subject, an effective amount of an armed oncolytic virus, e.g., an oncolytic virus encoding, in expressible form, an immunomodulator molecule, as described above.
  • an armed oncolytic virus e.g., an oncolytic virus encoding, in expressible form, an immunomodulator molecule
  • the administration of the oncolytic virus results in the secretion of the immunomodulator molecule from cells infected with the virus.
  • the immunomodulator molecule is IL-2.
  • the presently disclosed subject matter provides for methods of treating a subject suffering from a cancer, comprising administering, to the subject, an effective amount of an oncolytic virus encoding, in expressible form, a membrane-associated protein comprising an immunomodulator molecule linked to an anchoring peptide, as described above.
  • the immunomodulator molecule is IL-2.
  • the presently disclosed subject matter provides methods of inhibiting the growth and/or proliferation of and/or promoting the death of a cancer cell of a subject comprising administering to the subject a therapeutically effective amount of an oncolytic vaccinia virus described herein.
  • the presently disclosed subject matter provides methods of inhibiting the growth of a tumor of a subject comprising administering to the subject a therapeutically effective amount of an oncolytic vaccinia virus described herein.
  • the present invention further provides a method of reducing or inhibiting the growth of a tumor comprising administering to the tumor and/or contacting the tumor with a therapeutically effective amount of an oncolytic vaccinia virus described herein.
  • the presently disclosed subject matter provides methods for lengthening the period of survival of a subject having a cancer comprising administering to the subject a therapeutically effective amount of an oncolytic vaccinia virus described herein.
  • the period of survival of the subject having cancer is lengthened by about 1 month, about 2 months, about 4 months, about 6 months, about 8 months, about 10 months, about 12 months, about 14 months, about 18 months, about 20 months, about 2 years, about 3 years, about 5 years or more.
  • the presently disclosed subject matter further provides methods of treating a subject suffering from a cancer, comprising administering, to the subject, an effective amount of an oncolytic virus encoding, in expressible form, an immunomodulator molecule, which can be secreted from infected cells, and inducing the elicitation of potent antitumor T cells.
  • the presently disclosed subject matter also provides for methods of treating a subject suffering from a cancer, comprising administering, to the subject, an effective amount of an oncolytic virus encoding, in expressible form, a membrane-associated protein comprising an immunomodulator molecule linked to a heterologous anchoring peptide as described above, promoting the trafficking of the induced potent antitumor T cells into the tumor tissues where they would exert their cytotoxicity to cancer cells and associated stromal cells, thus displaying antitumor functions.
  • the presently disclosed subject matter also provides for methods of treating a subject suffering from a cancer, comprising administering, to the subject, an effective amount of an oncolytic virus encoding, in expressible form, a membrane-associated protein comprising an immunomodulator molecule linked to an anchoring peptide as described above, inducing the elicitation of potent antitumor T cells, and promoting the trafficking of the induced potent antitumor T cells into the tumor tissues where they would exert their cytotoxicity to cancer cells and associated stromal cells, thus displaying antitumor functions.
  • Certain non-limiting embodiments of the presently disclosed subject matter provide for methods of treating cancer as set forth above where the cancer is an early stage cancer. Certain non-limiting embodiments of the presently disclosed subject matter provide for methods of treating cancer as set forth above where the cancer is not an early stage cancer. Certain non-limiting embodiments of the presently disclosed subject matter provide for methods of treating cancer as set forth above where the cancer is a late stage cancer.
  • the presently disclosed subject matter provides for methods of treating cancer when there is increased tumor burden and/or edema in liver and/or kidneys, increased presence of immunosuppressive CD4 + Foxp3 + , CD4 + PD-1 + and CD8 + PD-1 + T cells, G-MDSC and PD-L1 + cells, and/or decreased presence of NK cells, or when there is increased PD-1, PD-L1, TGF- ⁇ and VEGF expression in the tumor microenvironment, compared with the early-stage cancer.
  • the administration of an effective amount of an oncolytic virus results in increased levels of IFN- ⁇ , Granzyme B, perforin and TGF- ⁇ , IL-10 and/or decreased levels of angiogenesis markers, e.g., CD105 and VEGF, in tumors that received an oncolytic virus encoding, in expressible form, a membrane-associated protein comprising an immunomodulator molecule linked to an anchoring peptide as described above, compared with tumors received the oncolytic virus encoding, in expressible form, a membrane-associated protein comprising an immunomodulator molecule that can be secreted.
  • angiogenesis markers e.g., CD105 and VEGF
  • the recently disclosed subject matter provides for methods of treating a subject suffering from a cancer, comprising administering, to the subject, an effective amount of an oncolytic virus, described herein, optionally in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody.
  • a checkpoint inhibitor e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody.
  • the recently disclosed subject matter provides for methods of treating a subject suffering from a cancer, comprising administering, to the subject, an effective amount of an oncolytic virus, described herein, optionally in combination with an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody.
  • a method disclosed herein can include administering, to the subject, an effective amount of an oncolytic virus that comprises a nucleic acid that encodes IL-2 in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • a method disclosed herein can include administering, to the subject, an effective amount of an oncolytic virus that comprises a nucleic acid that encodes IL-2 in combination with an anti-CTLA-4 antibody.
  • the method of treating a subject suffering from a cancer comprising administering, to the subject, an effective amount of a Western Reserve strain of vaccinia virus that comprises a nucleic acid that encodes IL-2, in combination with an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody.
  • the presently disclosed subject matter also provides for methods of treating a subject suffering from a cancer, comprising administering, to the subject, an effective amount of an oncolytic virus encoding, in expressible form, a membrane-associated protein comprising an immunomodulator molecule linked to an anchoring peptide as described above, in combination an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody.
  • a method disclosed herein can include administering, to the subject, an effective amount of an oncolytic virus that comprises a nucleic acid encoding a membrane-associated protein comprising IL-2 linked to an anchoring peptide in combination with an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • a method disclosed herein can include administering, to the subject, an effective amount of an oncolytic virus that comprises a nucleic acid encoding a membrane-associated protein comprising IL-2 linked to an anchoring peptide in combination with an anti-CTLA-4 antibody.
  • the method of treating a subject suffering from a cancer comprising administering, to the subject, an effective amount of a Western Reserve strain of vaccinia virus that comprises a nucleic acid that encodes IL-2 linked to an anchoring peptide, in combination with an anti-PD-1 antibody, an anti-PD-L1 antibody and/or an anti-CTLA-4 antibody.
  • the IL-2 is linked to the anchoring peptide via a rigid linker.
  • the oncolytic virus is a Western Reserve strain of vaccinia virus that comprises a nucleic acid that encodes IL-2 linked to an anchoring peptide via a rigid linker, e.g., a linker comprising the (A(EA 3 K) 4 AAA) linker.
  • methods of the presently disclosed subject matter can include depletion of Natural killer (NK) cells, CD8+ T cells and/or T cells.
  • methods of the presently disclosed subject matter can include neutralization of circulating IFN- ⁇ .
  • a method of the presently disclosed subject matter can include administering to a subject an oncolytic virus, as disclosed herein, and the depletion of CD8 + T cells, NK cells and/or CD4 + T within the subject and/or the neutralization of circulating IFN- ⁇ within the subject.
  • depletion and/or neutralization can be obtained by using antibodies, e.g., antibodies to CD8, CD4, NK1.1 and/or IFN- ⁇ .
  • a method for treating a subject suffering from a cancer can comprise administering, to the subject, an effective amount of an oncolytic virus that comprises a nucleic acid that encodes IL-2 and the deletion of NK cells.
  • the method can include administering, to the subject, an effective amount of a Western Reserve strain of vaccinia virus that comprises a nucleic acid that encodes IL-2 and the deletion of NK cells, e.g., by the administration of an anti-NK1.1 antibody.
  • a method for treating a subject suffering from a cancer can comprise administering, to the subject, an effective amount of an oncolytic virus that comprises a nucleic acid encoding a membrane-associated protein comprising an immunomodulator linked to an anchoring peptide and the deletion of NK cells.
  • a method disclosed herein can include administering, to the subject, an effective amount of an oncolytic virus that comprises a nucleic acid encoding a membrane-associated protein comprising IL-2 linked to an anchoring peptide and the deletion of NK cells.
  • the method can include administering, to the subject, an effective amount of a Western Reserve strain of vaccinia virus that comprises a nucleic acid that encodes a membrane-associated protein comprising IL-2 linked to an anchoring peptide and the deletion of NK cells, e.g., by the administration of an anti-NK1.1 antibody.
  • the subject suffers from an early stage cancer. In certain embodiments, the cancer is not an early stage cancer. In certain embodiments, the subject suffers from a late stage cancer.
  • compositions that include one or more isolated OV-induced T cells isolated from cancer tissue.
  • the present disclosure further provides pharmaceutical compositions comprising the modified viruses disclosed herein.
  • the pharmaceutical compositions containing a modified virus such as an oncolytic vaccinia virus, as described herein, can be prepared as solutions, dispersions in glycerol, liquid polyethylene glycols, and any combinations thereof in oils, in solid dosage forms, as inhalable dosage forms, as intranasal dosage forms, as liposomal formulations, dosage forms comprising nanoparticles, dosage forms comprising microparticles, polymeric dosage forms, or any combinations thereof.
  • compositions are formulated relative to the particular administration route.
  • pharmaceutical compositions that can be administered parenterally, intravenously, intradermally, intramuscularly, transdermally or intraperitoneally are described in U.S. Pat. Nos. 5,543,158, 5,641,515 and 5,399,363, the contents of which are incorporated by reference herein in their entireties.
  • a pharmaceutical composition as described herein can comprise a pharmaceutically acceptable carrier, e.g., an excipient.
  • a pharmaceutically acceptable carrier e.g., an excipient.
  • “Pharmaceutically acceptable,” as used herein, includes any carrier which does not interfere with the effectiveness of the biological activity of the active ingredients and/or that is not toxic to the patient to whom it is administered.
  • an excipient can be an excipient described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986).
  • Non-limiting examples of suitable excipients can include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a chelator, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener and a coloring agent.
  • an excipient can be a buffering agent.
  • suitable buffering agents can include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium glucomate, aluminium hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide and other calcium salts or combinations thereof can be used in a pharmaceutical formulation.
  • kits that comprise one or more of the disclosed oncolytic viruses described herein.
  • this disclosure provides for a kit for administering a modified virus as described herein.
  • a kit of this disclosure can include a modified virus or a pharmaceutical composition comprising a modified virus as described above.
  • this disclosure provides for a kit comprising one or more OV-induced T cells.
  • a kit of this disclosure can further include one or more components such as instructions for use, devices and additional reagents, and components, such as tubes, containers and syringes for performing the methods disclosed above.
  • a kit of this disclosure can include instructions for use, a device for administering the modified virus to a subject, or a device for administering an additional agent or compound to a subject.
  • the instructions can include a description of the modified virus and, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount and the proper administration method for administering the modified virus. Instructions can also include guidance for monitoring the subject over duration of the treatment time.
  • a kit of this disclosure can include a device for administering the modified virus to a subject.
  • a device for administering the modified virus can be included in the kits provided herein.
  • devices include, a hypodermic needle, an intravenous needle, a catheter, a needle-less injection device, an inhaler and a liquid dispenser, such as an eyedropper.
  • a modified virus to be delivered systemically can be included in a kit with a hypodermic needle and syringe.
  • this disclosure provides a kit for isolating tumor infiltrated T cells—induced by oncolytic virus (“OV-induced T cells”) from a subject with cancer after administering a modified virus as described herein.
  • OV-induced T cells induced by oncolytic virus
  • the present disclosure further provides kits that comprise isolated OV-induced T cells expanded according to the methods described herein.
  • the OV-induced T cells are cryogenically frozen.
  • the OV-induced T cells are provided in culture medium.
  • the kit can provide isolated T cells and further provide for dendritic cells and cancer cells, and/or one or more cytokines and/or one or more agents to treat T cells, including, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, IL-27, IFN-alpha, IFN-alpha2, IFN-beta, or IFN-gamma, TNF-alpha, TNF-beta, and GM-CSF.
  • kits of the present disclosure can include one or more additional agents that can be administered in combination with an oncolytic virus and/or isolated OV-induced T cells.
  • a kit can include a cytokine, e.g., IL-2, and/or an anti-PD-1 and/or an anti-PD-L1 antibody.
  • a kit of this disclosure can provide a device for isolating T cells from a subject after administering the modified virus to the subject, or a device for transferring the treated T cells into a subject.
  • a kit of this disclosure can provide instructions including a description of isolating OV-induced T cells and methods for transferring the treated T cells into a patient, including methods for determining the proper state of the subject, the proper dosage amount and the proper administration method for transferring the treated T cells.
  • a kit of this disclosure can include instructions for use, a device for administering the modified virus to a subject, and/or a device for administering an additional agent or compound to a subject, and/or a device for isolating the OV-induced T cells.
  • the instructions can include a description of the modified virus and, optionally, other components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount and the proper administration method for administering the modified virus.
  • Instructions can also include a description of isolating the OV-induced T cells, methods of treating the isolated T cells, and methods of transferring the treated T cells into a patient. Instructions can also include guidance for monitoring the subject over duration of the treatment time.
  • EXAMPLE 1 ONCOLYTIC VACCINIA VIRUS EXPRESSING MEMBRANE-ATTACHED IL-2 DISPLAYED POTENT ANTITUMOR EFFICACY AND REDUCED TOXICITY
  • vvDD oncolytic vaccinia virus
  • VVDD murine IL-2
  • mIL-2 murine IL-2
  • mice were inoculated subcutaneously on day 0, and then, on day 5, mice were administered either PBS or 1.0 e8 pfu of vvDD-IL-2 or, as a control, vvDD unarmed with mIL-2.
  • the number of mice in each test group was 10+/ ⁇ 4. Survival of the mice was monitored and the results are shown in FIGS. 1B-D ; in each case vvDD-mIL-2 prolonged survival substantially relative to the controls.
  • FIG. 2A when mIL-2-armed virus was administered nine days after MC38-luc (colon cancer) tumor cell inoculation, the recipient mice died within one week ( FIG. 2B ). As the tumor burden of these mice would be comparable to the control animals in this study, it is inferred that the animals died from toxic effects of secreted IL-2.
  • FIG. 3A shows a schematic diagram of a portion of the engineered vvDD virus, in which mIL-2 encoding nucleic acid, fused in-frame to nucleic acid encoding a (G45)3 linker and a GPI anchoring peptide having the sequence GPAGGGGSGGGGSGGGGSVSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTN I (SEQ ID NO:16), all operably linked to the p7.5 e/l promoter, is inserted in the tk gene to produce virus denoted “vvDD-mIL-2-GPI”.
  • YFP yellow fluorescent protein
  • vvDD-mIL-2-GPI expressed mIL-2 that was mostly retained on the membrane of infected cells, whereas mIL-2 expressed by vvDD-mIL-2 was not, as demonstrated by flow cytometry ( FIGS. 4A-B ).
  • the infected cells were gated by YFP and then analyzed for IL-2 expression on the gated YFP+ cells.
  • FIG. 5A-B presents the expression intensity of IL-2 on the cell membrane.
  • FIG. 6 shows the results of an experiment comparing “unarmed” vvDD (parental virus) with vvDD-mIL-2 and vvDD-mIL-2-GPI.
  • Mice were administered either PBS or (unarmed) vvDD, vvDD-mIL-2, or vvDD-mIL-2-GPI (at a dose of 1.0 e8 pfu) 5 days after tumor cell inoculation. As shown in FIG.
  • This Example provides studies of oncolytic vaccinia viruses expressing membrane-associated forms of immunostimulatory cytokines.
  • mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cancer cells and divided into required groups according to tumor growth condition based on live animal IVIS imaging 7 days post tumor cell injection.
  • Grouped mouse were intraperitoneally injected with 5 ⁇ 10 6 T cells per 100 ⁇ l or 100 ⁇ l PBS as a control.
  • T cells were generated by intratumoral vvDD-IL-2-GPI (1.0 e8 pfu) injection into MC38 s.c. tumor bearing mouse.
  • Ten days post virus-treatment tumors were harvested and T-cells were separated using CD90.2 beads (Miltenyl Biotec, CA, USA). T-cells were co-cultured with ⁇ DC-MC38 for 2 days.
  • FIG. 8A shows the timeline of the experimental setup.
  • oncolytic vaccinia viruses expressing novel forms of immunostimulatory cytokines can induce potent tumor-specific T cells, and promote the trafficking of such T cells into tumor tissues where they exert their functions against tumor growth.
  • OV-induced T cells can be isolated and expanded ex vivo under appropriate conditions. When these cultured and expanded tumor-specific T cells were infused back to a second tumor-bearing mice, they displayed potent anti-tumor activity in MC38 colon tumor model in syngeneic mice. The present approach demonstrated that oncolytic virus-elicited and enriched anti-tumor T cells can be utilized for cancer therapy.
  • Pleiotropic cytokine interleukin-2 (IL-2) is an established cancer therapeutic agent, whereas its clinical application has been limited for the severe life-threatening side effects followed by the systemic use of high dose of IL-2 1-4 .
  • IL-2 variants such as IL-2-fusion proteins, IL-2/anti-IL-2 antibody complexes and “superkines,” or chemical modified IL-2, to extend in vivo half-life and improve biological activity and safety 5-13 .
  • the present Example discloses a new form of IL-2 immunotherapy through local delivery of a cell membrane associated IL-2 in tumor bed by tumor-targeted oncolytic vaccinia virus.
  • IL-2 either flexibly or rigidly on the cell membrane cured mice with established early-stage peritoneal colon cancer, an effect similar to delivering secreted IL-2 by vaccinia virus. While secreted IL-2 delivered by vaccinia virus led to high mortality at the late-stage tumor model during the virus replication period, displaying IL-2 rigidly on cell membrane significantly enhanced the survival of mice with late-stage peritoneal colon cancer. Displaying IL-2 rigidly on the cell membrane led to profound changes in the immune status of tumor microenvironment, which made it feasible to combine anti-PD-1/PD-L1 Ab to cure most mice with late-stage peritoneal colon cancer. These findings show that the new form of IL-2 immunotherapy can be translatable to the clinic for cancer treatment.
  • Virus-delivered secreted IL-2 has been suggested to have the potential to treat established tumors in mouse models ( FIG. 1 ) 14 .
  • oncolytic vaccinia virus vvDD was used to reduce the severe toxic side effects caused by systemic use of high dose of IL-2 and to treat pleural, peritoneal, hematopoietic or metastatic cancer. Treating pleural, peritoneal, hematopoietic or metastatic cancer by intratumoural virus injection can be difficult.
  • oncolytic vaccinia virus which has been approved safe, to deliver membrane-associated IL-2 to treat peritoneal colon cancer, since membrane-associated cytokines have been suggested to retain on membrane without obvious compromised cytokine function 15-17 .
  • An approach using oncolytic vaccinia virus vvDD can result in the use of fewer viruses and can cause less side effects.
  • mice Female C57BL/6 (B6 in short) and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, Me.) and housed in specific pathogen-free conditions in the University of Pittsburgh animal facility. All animal studies were approved by the Institutional Animal Care and Use Committee. Mouse colon cancer MC38-luc, ovarian cancer ID8-luc, mesothelioma AB12-luc were generated as described previously 31 . Mouse melanoma B16 was obtained from ATCC.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • FBS penicillin/streptomycin
  • vSC20 a vgf gene-deleted Western Reserve (WR) strain VV was used as the parental virus for homologous recombination.
  • the plasmid pCMS1-IRES carrying two multiple cloning sites separated with an IRES sequence from pLVX-IRES-ZsGreen was constructed by us from the shuttle plasmid pSEM-1 32 .
  • pCMS1-IRES was then inserted with fragments containing flexible linker or rigid linker+GPI anchor sequence amplified form human CD16b by PCR, resulting plasmids pCMS1-IRES-FG or pCMS1-IRES-RG, respectively.
  • Murine IL-2 cDNA was inserted into pCMS1-IRES, pCMS1-IRES-FG or pCMS1-IRES-RG to get shuttle plasmids pCMS1-IL-2, pCMS1-IL-2-FG or pCMS1-IL-2-RG.
  • GPI anchor sequence in pCMS1-IL-2-FG was further replaced with murine PD-L1 transmembrane domain to get shuttle plasmid pCMS1-IL-2-FPTM. All these shuttle vectors were used for homologous recombination of murine IL-2 variants into the tk locus of vaccinia viral genome.
  • the primers for plasmid cloning based on PCR were listed in Table 2.
  • vvDD-IL-2 vvDD-IL-2-FG, vvDD-IL-2-RG and vvDD-IL-2-FPTM
  • CV-1 cells were infected with vSC20 at multiplicity of infection (MOI) of 0.1 and then transfected with the shuttle plasmids, resulting in viral seeds.
  • MOI multiplicity of infection
  • Selection of the new recombinant viruses was based on expression of yellow fluorescent protein in CV1 cells 24 h post seed virus infection.
  • vvDD-YFP, or vvDD in short a double viral gene-deleted (tk- and vgf-) VV carrying yfp cDNA at the tk locus, is the control virus for this work.
  • Tumor cells were seeded at 1.0 ⁇ 10 5 per well in six-well plates and infected with indicated viruses next day at MOIs of 0.1, 1.0, or 10 in 1 ml medium containing 2% fetal bovine serum for 2 hours. Following infection, cells were added 3 mL medium containing 10% fetal bovine serum and cultured until harvesting at 24, 48, and 72 hours post viral infection. The cell pellet was homogenized using a FastPrep Cell Disrupter (Model FP120; Qbiogene, Carlsbad, Calif.) to release virions, and the resulting cell lysates were titered on CV-1 cells to determine viral load by plaque assay.
  • a FastPrep Cell Disrupter Model FP120; Qbiogene, Carlsbad, Calif.
  • Tumor cells were plated at 1.0 ⁇ 10 4 cells per well in 96-well plates and infected with indicated viruses next day at MOIs of 0.05, 0.1, 0.5, 1.0, and 5.0. Cell viability was determined at 48 and 72 hours after infection, by CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay, or MTS assay (Promega, Madison, Mich.).
  • MC38-luc (3 ⁇ 10 5 ), B16 (2 ⁇ 10 5 ) or AB12-luc (3 ⁇ 10 5 ) cells were seeded in 24-well plates overnight and infected with vvDD, vvDD-IL-2, vvDD-IL-2-FG, vvDD-IL-2-RG or vvDD-IL-2-FPTM at MOI of 1 in 0.15 mL 2% FBS-containing-DMEM for 2 h. Cells were added 0.35 mL 10% FBS-containing-DMEM and cultured until harvesting at 24 h post viral infection.
  • the culture supernatants were harvested for measuring IL-2 by ELISA (BD Bioscience, San Jose, Calif.) and the cell pellets were applied either to measure membrane-associated IL-2 by flow cytometry, or to extract RNA to measure IL-2 expression by RT-qPCR.
  • mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc, 3.5 ⁇ 10 6 ID8-luc cancer cells or BALB/c mice were intraperitoneally inoculated with 4 ⁇ 10 5 AB12-luc, respectively, and divided into required groups 5 days or 9 days post tumor cell inoculation according to tumor size based on live animal IVIS imaging, performed using a Xenogen IVIS 200 Optical In Vivo Imaging System (Caliper Life Sciences, Hopkinton, Mass.). Grouped mice were intraperitoneally injected with indicated viruses, antibodies, the combinations or PBS, respectively.
  • ⁇ -CD8 Ab at 250 ⁇ g/injection (clone 53-6.7; Bio X Cell), ⁇ -CD4 Ab (clone GK1.5, Bio X Cell; 150 ⁇ g/injection), ⁇ -NK1.1 (clone PK136, Bio X Cell; 300 ⁇ g/injection) or ⁇ -IFN- ⁇ Ab (clone XMG1.2, Bio X Cell; 200 ⁇ g/injection) were intraperitoneally injected into mice to deplete CD8 + T cells, CD4 + T cells, NK1.1 + cells or neutralize circulating IFN- ⁇ . In some experiments, mice were sacrificed to harvest tumor tissues and spleens at indicated time points.
  • Virus-treated mice were sacrificed 4 to 5 days post treatments for collection of blood, lungs, kidneys and spleens. Blood samples were kept for 2 hours at room temperature and separated sera by centrifugation for measuring IL-2 and TNF- ⁇ using commercialized kits (BD Biosciences and BioLegend, respectively), according to the vendors' instructions. Water content was used to monitor tissue edema. Briefly, wet tissue was weighed, and dehydrated overnight at 90° C. in chemical hood. The weight difference between wet tissues and dry tissues was calculated.
  • RPMI 1640 medium containing 2% FBS, 1 mg/ml collagenase, 0.1 mg hyaluronidase, and 200 U DNase I (All enzymes were from Sigma, St. Louis, Mo.) at 37° C. for 1-2 h to make single cells.
  • RNA was extracted from virus-infected cells or tumor tissues using the RNeasy Kit (Qiagen, Valencia, Calif.). One microgram of RNA was used for cDNA synthesis, and 25 to 50 ng of subsequent cDNA was used to conduct mRNA expression analysis by TaqMan analysis on the StepOnePlus system (Life Technologies, Grand Island, N.Y.). All the primers for the analysis were purchased from Thermo Fisher Scientific (Waltham, Mass.). The gene expression was normalized to a house-keeping gene HPRT1 and expressed as fold increase (2 ⁇ CT ), where ⁇ CT CT (Target gene) ⁇ CT (HPRT1) .
  • Viruses vvDD-IL-2, vvDD-IL-2-FPTM, vvDD-IL-2-FG and vvDD-IL-2-RG were generated based on vvDD ( FIG. 9 ).
  • vvDD-IL-2 produced secreted murine IL-2 and vvDD-IL-2-FPTM
  • vvDD-IL-2-FG and vvDD-IL-2-RG displayed murine IL-2 on cell membrane post infection.
  • vvDD-IL-2-FPTM produced IL-2 fused with murine PD-L1 transmembrane domain and a flexible linker (G 4 S) 3 in between.
  • vvDD-IL-2-FG and vvDD-IL-2-RG produced IL-2 fused with glycoinositol phospholipid (GPI) anchor sequence of human CD16b, and a flexible linker (G 4 S) 3 and a rigid linker (A(EA 3 K) 4 AAA) in between, respectively.
  • GPI glycoinositol phospholipid
  • A(EA 3 K) 4 AAA rigid linker
  • vvDD-IL-2 produced significantly more IL-2 in supernatant than others, but not membrane associated IL-2.
  • vvDD-IL-2-FPTM, vvDD-IL-2-FG and vvDD-IL-2-RG produced few IL-2 in supernatant, but significantly more membrane-associated IL-2 than VVDD-IL-2.
  • the amount of membrane associated IL-2 in vvDD-IL-2-RG was more than vvDD-IL-2-FG and the amount of membrane associated IL-2 in vvDD-IL-2-FG was more than vvDD-IL-2-FPTM ( FIG. 11A ).
  • IL-2 and viral marker gene A34R were determined in tumor cell post virus infection in vitro.
  • the data showed that viral gene A34R mRNA were similar, but IL-2 mRNA pattern was similar to the pattern of the membrane associated IL-2 amount described above. Without being bound to a particular theory, this shows that the precise component of chimeric proteins can impact the mRNA stability and further impact the amount of IL-2 displaying on cell membrane ( FIG. 12 ).
  • each virus was injected intraperitoneally at the dose of 2 ⁇ 10 8 PFU/mouse to treat B6 mice received murine colon cancer cell MC38-luc inoculation 5 days ago (early-stage tumor model).
  • the survival results showed that vvDD-IL-2, vvDD-IL-2-FG and vvDD-IL-2-RG, but not vvDD-IL-2-FPTM, elicited more potent antitumor effects compared with PBS or vvDD ( FIG. 11B ).
  • this can be explained by the low amount of IL-2 displayed on cell membrane post vvDD-IL-2-FPTM infection ( FIG. 11A and FIG. 12 ).
  • the mice treated with vvDD-IL-2 survived longer than those treated with vvDD-IL-2-FG, but not than those treated with vvDD-IL-2-RG ( FIG. 11B ).
  • mice that survived the vvDD-IL-2, vvDD-IL-2-FG and vvDD-IL-2-RG treatment rejected a subcutaneous tumor rechallenge, which showed that a systemic antitumor response was elicited ( FIG. 13 ).
  • the late-stage tumor mice also have increased severe edema in the livers and kidneys ( FIG. 15 ), compared with the early-stage tumor model.
  • the therapeutic results from the late-stage tumor model showed that the vvDD-IL-2 treatment led to a high mortality within one-week post treatment, whereas treatment with the other viruses were safe.
  • the vvDD-IL-2-FG and vvDD-IL-2-RG treatments significantly extended the animal survival, compared with the vvDD treatment, however, the vvDD-IL-2-RG treatment elicited a significantly better survival ( FIG. 11C and FIG. 16A ).
  • IL-2 serum levels were measured and it was found that they were 100 times higher in the serum of mice treated with vvDD-IL-2, compared to the serum of mice treated with the other viruses and reached about 24950 pg/mL ( FIG. 16B ). Low levels of IL-2 were also detected in the serum of mice treated with vvDD-IL-2-FG and vvDD-IL-2-RG.
  • TNF- ⁇ serum levels were measured after vvDD-IL-2 treatment and it was found that vvDD-IL-2 treatment induced a significant increased in the TNF- ⁇ serum levels ( FIG. 16C ). Mice were also assessed for tissue edema, a hallmark for measuring IL-2 induced vascular leak syndrome 20 . Only vvDD-IL-2 treatment induced substantial more pulmonary and hepatic edema, as evidenced by the water content increase in lungs and livers ( FIGS. 16D-16E ). Tissue edema was also found.
  • CD4 + Foxp3 + T cells were examined.
  • the percentages of CD4 + T regulatory cells in tumors were similar to the ones described above ( FIG. 18D ).
  • the CD8 + /Treg ratio in tumors that received vvDD-IL-2-RG treatment was significantly higher, since CD8 + T cells were elevated in tumor post vvDD-IL-2-RG treatment compared to tumors that received other virus treatment ( FIG. 18E and FIG. 19D ).
  • IL-10 can function as both immune stimulation and immune suppression in cancer, here IL-10 can have an inhibitory effect on the expression TNF- ⁇ , which was elevated by IL-2 and recently suggested as a tumor growth factor for minimal residual tumor, thereby making VVDD-IL-2-RG treatment safer and more effective ( FIG. 16C ) 19,22-24 .
  • the reason could be that the NK cells could impede virotherapy via the natural cytotoxicity receptor NKP46.
  • the expression of NKP46 was significantly increased by the vvDD-IL-2-RG treatment, thus, NK cell depletion could have removed this impediment ( FIG. 20 ) 25 .
  • vvDD-IL-2-FG and vvDD-IL-2-RG were tested to investigate whether the combination could treat 9-day-tumour-bearing mice.
  • the results showed that vvDD-IL-2-RG combined with an anti-PD-1/PD-L1 antibody, but not with an anti-CTLA-4 antibody, cured tumor-bearing mice ( FIG. 22D ). Without being bound to a particular theory, this could be attributed to the different mechanisms for anti-CTLA-4 and anti-PD-1/PD-L1 checkpoint blockade.
  • Anti-CTLA-4 antibodies primarily affect CD4 + T cells at prime phase, while anti-PD-1/PD-L1 antibodies predominantly act on exhausted T cells within the tumor, which could important for overcoming the more immunosuppressive microenvironment in late-stage large solid tumors.
  • the combination of VVDD-IL-2-FG with anti-PD-1/PD-L1 antibody did not improve the survival. Without being bound to a particular theory, this suggests that changes in immune status in tumor-bearing mice, especially in late-stage-tumor-bearing mice, was essential for the effectiveness of the monotherapy and the combination therapy. This could be evidenced by several recent reports showing the effectiveness of oncolytic viruses combined with immune checkpoint blockade in preclinical models and clinical trails ( FIG. 22D and FIG. 24 ) 27-30 .
  • the present Example demonstrated that vvDD-IL-2-RG treatment could effectively elicit antitumor immune status change in late-stage-tumor-bearing mice with significantly reduced systemic toxicity which was induced by IL-2 application, and finally led to a much better survival.
  • the combination of vvDD-IL-2-RG with anti-PD-1 and PD-L1 antibodies can cure mice with late-stage-tumor.
  • the present Example showed that vvDD-IL-2-RG treatment can be a new form of IL-2 immunotherapy that can be translatable to the clinic for cancers and immunosuppressive cancers.
  • Immunotherapy is rapidly evolving and fighting cancer by re-activating the patient immune system presents an attractive therapeutic strategy for gastrointestinal cancer besides standard treatments as surgery, chemotherapy and radiotherapy.
  • Gastrointestinal cancer including colorectal, gastric, liver and biliopancreatic cancer belong to the ten most frequent malignancies worldwide (1).
  • Immune infiltration impacts tumor progression and patient survival and that a strong lymphocyte infiltration has been reported to be associated with an antitumor response and improved clinical outcome in several GI adenocarcinomas (2-9).
  • Different immunotherapeutic strategies as cancer vaccines adoptive T cell transfer of autologous T cells or checkpoint blockade have been developed but are still not available for a wide range of GI tumors due to tumor heterogeneity and poor immune infiltration.
  • Tumor cells develop immune escape mechanism modulating the immune system to avoid detection by effector cells.
  • immune system checkpoint ligands such as programmed death ligand 1(PD-L1) (10,11), secretion of soluble immunosuppressive factors such as transforming growth factor beta (TGF-B), vascular endothelian growth factor (VEGF), interleukin-10 (Il-10), galectin-1, indoleamine 2,3-dehydrogenase (12-14), and down-regulation of major histocompatibility complex (MHC) class I expression; overexpression of receptors such as C—X—C chemokine receptor type 4 (CXCR4), basic fibroblast growth factor and epidermal growth factor (15,16).
  • PD-1 programmed death ligand 1
  • TGF-B vascular endothelian growth factor
  • Il-10 interleukin-10
  • galectin-1 galectin-1
  • indoleamine 2,3-dehydrogenase (12-14) and down-regulation of major histocompatibility complex
  • the immunosuppressive tumor microenvironment presents optimal conditions for the recruitment of immunosuppressive macrophages, MDSCs and Tregs interfering with an efficient anti-tumor T cell response.
  • Inhibitory checkpoint molecules as CTL-4, PD-1, TIM3, LAG3, upregulated in chronically stimulated T cells, promoting even further T cell anergy.
  • TEE immunosuppressive tumor microenvironment
  • a strategy to reverse the immunosuppressive tumor microenvironment (TME) and break immune tolerance is presented by oncolytic virotherapy. After selectively infecting and replicating in cancer cells and associated endothelial cells they kill these cells in cancerous tissue while leaving unaffected, healthy tissue unharmed (17,18).
  • Immunogenic cell death (ICD) of stromal cells and cancer cells induced by OV exposes a natural repertoire of tumor-associated antigens (TAAs) in conjunction with danger signals (damage associated molecular pattern (DAMPs)) and OV-derived pathogen-associated molecular pattern (PAMP) molecules and inflammatory cytokines to elicit anti-tumor immunity (19-21).
  • TAAs tumor-associated antigens
  • DAMPs damage associated molecular pattern
  • PAMP OV-derived pathogen-associated molecular pattern
  • an oncolytic vaccinia virus (Western Reserve Strain) vvDD was shown to be tumor selective and to promote anti-tumor response (23,24).
  • various oncolytic VV were engineered to express tumor antigens, T-cell co-stimulatory molecules and inflammatory cytokines (25). Their efficacy and safety have been demonstrated in preclinical studies (26-30).
  • the present Example showed for the first time that oncolytic vaccinia virus-induced tumor infiltrating T cells (0V-induced T cells) (also referred to herein as “tumor-infiltrated T cells” or “TILs”) can be used for ex vivo expansion and adoptive T cell transfer as new immunotherapeutic approach.
  • mice Female C57BL/6 mice were obtained from The Jackson Laboratory (Bar, Harbor, Me., USA) and housed in specific pathogen-free conditions in the University of Pittsburgh animal facility. All animal studies were approved by the Institutional Animal Care and Use Committee of the University.
  • Murine colon cancer cell lines MC38 and B16 were obtained from ATCC. Mouse colon cancer MC38-luc was generated as described previously. All cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1 ⁇ penicillin/streptomycin solution (Invitrogen, Carlsbad, Calif.) in 37° C., 5% CO2 incubator.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • 2 mM L-glutamine 1 ⁇ penicillin/streptomycin solution
  • vaccinia virus (Western Reserve strain) vvDD, vvDD-CCL5 (31) and vvDD-CXC11 (32) were previously described. In the presently disclosed subject matter newly developed vvDD-IL-15 and vvDD-IL-2 are also provided. Virus has been purified in HeLa cells. Virus titer has been defined in CV-1 cells using plaque assay. For intratumoral virus application 1 e8 pfu per mouse have been used.
  • mice were subcutaneously inoculated with 5 ⁇ 10 5 MC38 cancer cells.
  • vvDD-IL-2, vvDD-IL-15, vvDD-CXC11, vvDD-CCL5, vvDD or PBS was intratumorally injected at 1 e8 pfu/tumor when the s.c. tumor area reached 5 ⁇ 5 mm 2 .
  • IL-2 was administered intratumoral with 1 ⁇ 10 6 IU per tumor (Prometheus, San Diego, Calif.). The primary tumor size was measured using an electric caliper in two perpendicular diameters followed by every other day measurement.
  • mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cells and randomized according to tumor size based on live animal IVIS imaging 7 days post tumor cell injection using a Xenogen IVIS 200 Optival In Vivo Imaging System (Caliber Life Sciences, Hoptikon, Mass.).
  • B6 mice were s.c. inoculated with 5 ⁇ 10 5 MC38 cancer cells.
  • 5 ⁇ 5 mm 2 vvDD-IL-2 (1 e 8 pfu per tumor) was intratumorally injected.
  • 10 days later tumors have been collected and were incubated at 37° C. in digestion buffer (Miltenyl Biotec, San Diego, Calif.) before being mashed over a 100 ⁇ M tissue strainer. Lysis of red blood cell was performed using ACK Lysing buffer (Thermo Fisher Scientific, Waltham, Mass.).
  • T cells have been cultured in 24-well plates in a concentration with 1 ⁇ 10 6 per well in RPMI complete media with 30 IU/ml IL-2 (Miltenyl Biotec, San Diego, Calif.) and 5 ng/ml IL-7 (Biolegend, San Diego, Calif.) over 4 days.
  • As control T cells spleens from non-tumor bearing untreated mouse have been harvested and proceed to single cell suspension followed by magnetic separation (CD90.2 beads).
  • the na ⁇ ve T cells have been cultured under the same conditions as the virus induced T cells.
  • T cells Prior to adoptive T cell transfer, T cells have been analyzed for tumor specificity in a co-culture assay.
  • T cells (2 ⁇ 10 4 per well) were either left unstimulated (medium) or challenged with ⁇ -irradiated MC38 tumor cells (2 ⁇ 10 4 per well, 96-well plate) or irrelevant target cells as ⁇ -irradiated B16 tumor cells (2 ⁇ 10 4 per well) or na ⁇ ve splenocytes (2 ⁇ 10 4 per well) from non-tumor-bearing B6 mouse in duplicate for 24 h.
  • the plate setup has been used for IFN- ⁇ ELISPOT or Flow cytometry analysis as described above.
  • mice were intraperitoneally inoculated with 5 ⁇ 10 5 MC38-luc cancer cells and divided into required groups according to tumor growth condition based on live animal IVIS imaging 7 days post tumor cell injection.
  • Grouped mouse received 1 ⁇ 10 6 vvDD-IL-2 induced T cells, na ⁇ ve T cells or PBS.
  • All treatment mouse received 5 Gy of sublethal irradiation to mimic lymphodepletion according to clinical protocols prior to cell transfer and exogenous cytokine support of IL-2 (100.000 IU/mouse i.p. for 3 days post transfer every 12 h) (Prometheus, San Diego, Calif.).
  • Post 24 h co-culture assay was performed as described above. T cells were stained using Zombi aqua (Biolegend, San Diego, Calif.) followed by staining with antibodies against mouse CD3, CD8, CD4, 4-1BB (Biolegend, San Diego, Calif.). Samples were collected by BD Bioscience LSRII Fortessa. Data were analyzed by BD FACS Diva software and FlowJo software (Tree Star Inc., Ashland, Oreg.).
  • T cells were either left unstimulated (medium) or challenged with ⁇ -irradiated MC38 tumor cells (2 ⁇ 10 4 per well, 96-well plate) or irrelevant target cells as ⁇ -irradiated B16 tumor cells (2 ⁇ 10 4 per well) or na ⁇ ve splenocytes (2 ⁇ 10 4 per well) from non-tumor-bearing B6 mouse in duplicate for 24 h.
  • biotylated secondary antibody (clone R4-6A2-biotin, Mabtech, Inc) was added and incubated at room temperature for 2 h. Spot development followed using Vectastain Elite ABC and AEC Peroxidase substrate (SK-4200) kits (Vector Laboratories, Inc. Burlingame, Calif.). Number of IFN- ⁇ spots were analyzed by ImmunoSpotTM (Cellular Technology, Ltd., Shaker Heights, Ohio).
  • mice The health and survival of treated mice was closely monitored. All mice bearing subcutaneous or peritoneal tumors were monitored via caliper measurements for changes in tumor size or abdominal girth. Mice died naturally due to the disease or sacrificed when their subcutaneous tumor size exceeded 20 mm in diameter or when abdominal girth exceeded 1.5 ⁇ the original measurement.
  • vvDD-IL-2 is a Potent Modulator of the TME Attracting High Numbers of Tumor Reactive T Cells.
  • the T cell immune response in the TME of MC38 s.c. tumor bearing mouse was tested 10 days post intratumoral virus treatment with vvDD, vvDD-CXC11, vvDD-CCL5, vvDD-IL-15, vvDD-IL-2 or PBS.
  • CD8+ T cells from virus-treated tumors were analyzed for IFN- ⁇ response by ELISPOT.
  • ELISPOT ELISPOT
  • all virus-treated tumors presented a significant increase in reactive CD8+ T cells when tested against ⁇ -irradiated MC38 tumor cells.
  • vvDD-IL-2 treated tumors presented the highest level of reactive CD8+ T cells compared to vvDD or vvDD-IL-15 treated tumors ( FIG. 25A ).
  • CD8+ T cells are tumor specific.
  • IFN- ⁇ response of CD8+ T cells from vvDD-IL-2, vvDD or PBS treated tumors was measured post co-culture with irrelevant target cells, e.g., ⁇ -irradiated B16 tumor cells, na ⁇ ve splenocytes from non-tumor-bearing B6 mouse and ⁇ -irradiated MC38 tumor cells.
  • Virus-treated tumors presented a significant increase in MC38-reactive CD8+ T cells compared to PBS control.
  • vvDD-IL-2 induced CD8+ T cells exhibited highly specific reactivity against MC38 tumor cells compared to irrelevant target cells, e.g., B16 and na ⁇ ve splenocytes.
  • the vvDD induced T cells presented similar levels of MC38 reactivity compared to vvDD-IL-2, but exhibited higher background reactivity against the unspecific control cells as B16 and na ⁇ ve splenocytes compared to vvDD-IL-2 ( FIG. 25B ).
  • the vvDD-IL-2 induced T cells are highly MC38 specific.
  • Virally Expressed IL-2 Promotes a Strong Infiltration of Tumor Reactive CD8+ T Cells in the TME Compared to Single IL-2 Treatment.
  • MC38 s.c. tumor bearing mouse were treated intratumorally with IL-2, vvDD-IL-2, vvDD or PBS. 10 days later, tumor infiltrating CD8+ T cells were analyzed for IFN- ⁇ response by ELISPOT. The IFN- ⁇ response of vvDD-IL-2 induced CD8+ T cells was prior compared to IL-2 treatment, control virus or PBS ( FIG. 26 ).
  • vvDD-IL-2 Promotes T Cell Infiltration in the ME of MC38 Tumor Bearing Mouse.
  • FIGS. 27 and 28 To quantify the ability of vvDD-IL-2 to promote T cell infiltration in the TME, treated tumors were analyzed by immunofluorescence staining for total CD3+ infiltrating cells and CD3+CD8+ and CD3+CD4+ T cells ( FIGS. 27 and 28 ). Both virus constructs promote significant increase of total CD3+ cells and CD8+ T cell infiltration in the TME compared to PBS control. Virus treatment presented an increase in CD4+ T cell infiltration compared to PBS samples. Similar results were observed in other experimental setups, when T cells were purified from tumor tissue and quantified per gram tumor ( FIG. 28 ).
  • Vvdd-IL-2 Induced Tumor Infiltrating t Cells can be Expanded and Keep their Tumor specificity.
  • the T cells were tested for their tumor recognition using a co-culture assay, including relevant target cells (irradiated MC38 tumor cells) and irrelevant target cells as ⁇ -irradiated B16 tumor cells or na ⁇ ve splenocytes from non-tumor-bearing B6 mouse. After 24 h, the cells were analyzed for tumor specificity using IFN- ⁇ ELISPOT or 4-1BB expression by flow cytometry.
  • T cells from vvDD-IL-2, vvDD or PBS treated tumors presented tumor specific IFN- ⁇ secretion compared to irrelevant target cells, e.g., B16 or na ⁇ ve splenocytes ( FIG. 29B ).
  • FIG. 29C vvDD-IL-2 induced T cells presented highly tumor specific CD8+ and CD4+ T cells with less unspecific reactivity against irrelevant target cells when compared to vvDD or PBS group ( FIG. 29C ).
  • mice Prior to T cell transfer, treated mouse received 5 Gy of sublethal irradiation to mimic lymphodepletion similar to clinical protocols. Grouped mouse were intraperitoneally injected with vvDD-IL-2-induced T cells, na ⁇ ve T cells or PBS. All treated mouse received exogenous cytokine support of IL-2. The therapeutic response was monitored by live animal bioluminescence imaging to monitor the kinetics of tumor growth over time ( FIG. 33C ). Mouse that received vvDD-IL-2 induced T cells presented the strongest tumor regression compared to control mouse.
  • the virus-generated T cells led to the best overall survival compared to mice that received na ⁇ ve T cells or only irradiation with IL-2 or PBS ( FIG. 33C ). Imaging Day 17 post ⁇ CT is shown in FIG. 34 .
  • FIG. 31 Representative flow cytometry plot of CD8 + 4-1BB + and CD4 + 4-1BB + T cells of one sample from each group is shown in FIG. 32 .
  • T cells were analyzed for 4-1BB expression by flow cytometry and IFN- ⁇ secretion by ELISPOT.
  • Virus derived T cells presented tumor specific IFN- ⁇ spots with nearly no reactivity against irrelevant target cells compared to na ⁇ ve T cells ( FIGS. 31B AND 31C ).
  • FIGS. 31B AND 31C When the T cells from the same samples as used for ELISPOT were analyzed for 4-1BB expression, only the virus induced T cells presented significant tumor specific CD8+4-1BB+ and CD4+4-1BB expression when compared to control group.
  • the present Example shows that cytokine-armed oncolytic vaccinia virus can promote intratumoral T cell infiltration and generates tumor specific T cells (0V-induced T cells) for adoptive T cell transfer.
  • Intratumoral application of cytokine-armed oncolytic vaccinia virus promotes T cell infiltration into the TME.
  • the virus induced T cells were highly tumor specific and kept their therapeutic potential when expanded ex vivo and transferred into tumor bearing mouse.
  • the present Example presents a strategy to promote intratumoral T cell infiltration and to generate tumor specific T cells in the tumor microenvironment for adoptive T cell transfer.

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